Perhydrolase

ABSTRACT

The present invention provides methods and compositions comprising at least one perhydrolase enzyme for cleaning and other applications. In some particularly preferred embodiments, the present invention provides methods and compositions for generation of peracids. The present invention finds particular use in applications involving cleaning, bleaching and disinfecting.

The present application is a continuation application of U.S.application Ser. No. 10/581,014, filed Sep. 11, 2007, now U.S. Pat. No.8,772,007, which is a U.S. National Stage application of InternationalApplication No. PCT/US04/40438, filed Dec. 3, 2004, which claimspriority under 35 U.S.C. §119 to U.S. Provisional Patent ApplicationSer. No. 60/526,764, filed Dec. 3, 2003, which are hereby incorporatedherein in their entireties.

FIELD OF THE INVENTION

The present invention provides methods and compositions comprising atleast one perhydrolase enzyme for cleaning and other applications. Insome particularly preferred embodiments, the present invention providesmethods and compositions for generation of peracids. The presentinvention finds particular use in applications involving cleaning,bleaching and disinfecting.

BACKGROUND OF THE INVENTION

Detergent and other cleaning compositions typically include a complexcombination of active ingredients. For example, most cleaning productsinclude a surfactant system, enzymes for cleaning, bleaching agents,builders, suds suppressors, soil-suspending agents, soil-release agents,optical brighteners, softening agents, dispersants, dye transferinhibition compounds, abrasives, bactericides, and perfumes. Despite thecomplexity of current detergents, there are many stains that aredifficult to completely remove. Furthermore, there is often residuebuild-up, which results in discoloration (e.g., yellowing) anddiminished aesthetics due to incomplete cleaning. These problems arecompounded by the increased use of low (e.g., cold water) washtemperatures and shorter washing cycles. Moreover, many stains arecomposed of complex mixtures of fibrous material, mainly incorporatingcarbohydrates and carbohydrate derivatives, fiber, and cell wallcomponents (e.g., plant material, wood, mud/clay based soil, and fruit).These stains present difficult challenges to the formulation and use ofcleaning compositions.

In addition, colored garments tend to wear and show appearance losses. Aportion of this color loss is due to abrasion in the laundering process,particularly in automated washing and drying machines. Moreover, tensilestrength loss of fabric appears to be an unavoidable result ofmechanical and chemical action due to use, wearing, and/or washing anddrying. Thus, a means to efficiently and effectively wash coloredgarments so that these appearance losses are minimized is needed.

Cleaning compositions that comprise esterases, lipases and cutinases arewell-known in the art. However, these enzymes have a very low ratio ofperhydrolysis to hydrolysis. This results in the conversion of most ofthe ester substrate into acid, instead of the more desirable peracid.This is a serious drawback, since formula space and cost considerationsrender it feasible to include only a limited amount of substrate.

In sum, despite improvements in the capabilities of cleaningcompositions, there remains a need in the art for detergents that removestains, maintain fabric color and appearance, and prevent dye transfer.In addition, there remains a need for detergent and/or fabric carecompositions that provide and/or restore tensile strength, as well asprovide anti-wrinkle, anti-bobbling, and/or anti-shrinkage properties tofabrics, as well as provide static control, fabric softness, maintainthe desired color appearance, and fabric anti-wear properties andbenefits. In particular, there remains a need for the inclusion ofcompositions that are capable of removing the colored components ofstains, which often remain attached to the fabric being laundered. Inaddition, there remains a need for improved methods and compositionssuitable for textile bleaching.

In addition to the fabric and garment cleaning area, bleaching iscommonly used in the pulp and paper industry. Prior to production ofpaper, pulp is typically treated to remove undesirable coloredcontaminants. This provides pulp that is suitable for production ofpaper of higher quality than pulp that is not treated to remove coloredcontaminants and other undesirable components present in pulp. Forexample, in the paper recycling industry, removal of ink is necessary.Although standard methods are suitable for deinking paper with oil orwater-based inks, the increased use of electrostatic inks has madedeinking problematic, as these inks are much more difficult to remove.There are various methods available for deinking paper, including theuse of enzymes (See e.g., U.S. Pat. No. 5,370,770). However, thereremains a need in the art for efficient, cost-effective methods fortreatment of pulp for paper (recycled and new) product production.

Bleaching is also commonly used in the personal care market (e.g.,dental whiteners, hair bleachers, etc.). Although personal carebleaching products have improved over the years, there remains a needfor mild, easy to use, cost-effective bleaching methods for thissetting.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions comprising atleast one perhydrolase enzyme for cleaning and other applications. Insome particularly preferred embodiments, the present invention providesmethods and compositions for generation of peracids. The presentinvention finds particular use in applications involving cleaning,bleaching and disinfecting.

In some embodiments, the present invention provides compositionscomprising at least one perhydrolase, wherein the perhydrolase exhibitsa perhydrolysis to hydrolysis ratio that is greater than 1.

The present invention also provides isolated perhydrolases, wherein theperhydrolases exhibit a perhydrolysis to hydrolysis ratio that isgreater than 1. In some preferred embodiments, the perhydrolase is M.smegmatis perhydrolase. In alternative preferred embodiments, theperhydrolase is at least approximately about 35% homologous to M.smegmatis perhydrolase. In further embodiments, the perhydrolase is atleast approximately about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 99% homologous to M. smegmatis perhydrolase. Inadditional preferred embodiments, the perhydrolase comprises the aminoacid sequence set forth in SEQ ID NO:2. In some preferred embodiments,the perhydrolases have immunological cross-reactivity with M. smegmatisperhydrolase. In still further embodiments, the perhydrolase is at leasta portion of M. smegmatis perhydrolase, wherein the perhydrolase has aperhydrolysis to hydrolysis ration that is greater than 1. Inalternative embodiments, the perhydrolase is a structural homologue ofM. smegmatis perhydrolase, in which the active site is homologous to atleast one amino acid selected from the group consisting of S11, D192,and H195 of the M. smegmatis perhydrolase.

The present invention also provides isolated perhydrolase variantshaving amino acid sequences comprising at least one modification of anamino acid made at a position equivalent to a position in M. smegmatisperhydrolase comprising the amino acid sequence set forth in SEQ IDNO:2. In some embodiments, at least one modification is made at an aminoacid position equivalent to a position in M. smegmatis perhydrolasecomprising the amino acid sequence set forth in SEQ ID NO:2, wherein themodified amino acid is selected from the group consisting of Cys7,Asp10, Ser11, Leu12, Thr13, Trp14, Trp16, Pro24, Thr25, Leu53, Ser54,Ala55, Thr64, Asp65, Arg67, Cys77, Thr91, Asn94, Asp95, Tyr99, Val125,Pro138, Leu140, Pro146, Pro148, Trp149, Phe150, Ile153, Phe154, Thr159,Thr186, Ile192, Ile194, and Phe196. In further embodiments, themodification comprises at least one substitution at an amino acidposition equivalent to a position in M. smegmatis perhydrolasecomprising the amino acid sequence set forth in SEQ ID NO:2, wherein atleast one substitution is selected from the group consisting of M1, K3,R4, I5, L6, C7, D10, S11, L12, T13, W14, W16, G15, V17, P18, V19, D21,G22, A23, P24, T25, E26, R27, F28, A29, P30, D31, V32, R33, W34, T35,G36, L38, Q40, Q41, D45, L42, G43, A44, F46, E47, V48, I49, E50, E51,G52, L53, S54, A55, R56, T57, T58, N59, I60, D61, D62, P63, T64, D65,P66, R67, L68, N69, G70, A71, S72, Y73, S76, C77, L78, A79, T80, L82,P83, L84, D85, L86, V87, N94, D95, T96, K97, Y99F100, R101, R102, P104,L105, D106, I107, A108, L109, G110, M111, S112, V113, L114, V115, T116,Q117, V118, L119, T120, S121, A122, G124, V125, G126, T127, T128, Y129,P146, P148, W149, F150, I153, F154, I194, and F196.

In some preferred embodiments, the variant perhydrolase exhibits achange in peracid hydrolysis compared to the wild-type perhydrolase. Insome embodiments, the change in peracid hydrolysis is a decrease, whilein other embodiments, the change in peracid hydrolysis is an increase.

In some alternative preferred embodiments, the variant perhydrolaseexhibits a ratio of peracid hydrolysis of about 0.1 or less, incomparison with wild-type perhydrolase. In alternative preferredembodiments, the variant perhydrolase comprises at least onemodification comprising at least one substitution at an amino acidposition equivalent to a position in M. smegmatis perhydrolasecomprising the amino acid sequence set forth in SEQ ID NO:2, wherein atleast one substitution is selected from the group consisting of R4, L12,G15, P18, R27, W34L38, A44, E51, G52, L53, S54, T58, R67, L68, S72, A79,T80, D85, L86, V87, N94, K97, R101, V118, L119, G124, G126, and I194.

In further alternative embodiments, the variant perhydrolase exhibits aratio of peracid hydrolysis of about 0.2 or less, in comparison withwild-type perhydrolase. In yet additional embodiments, the variantperhydrolase comprises at least one modification comprising at least onesubstitution at an amino acid position equivalent to a position in M.smegmatis perhydrolase comprising the amino acid sequence set forth inSEQ ID NO:2, wherein at least one substitution is selected from thegroup consisting of R4, I5, D10, L12, W14, G15, P18, V19, T25, R27, W34,L38, A44, I49, E50, E51, G52, L53, S54, A55, R56, T58, N59, D62, T64,D65, R67, L68, N69, S72, S76, C77, A79, T80, L82, P83, D85, L86, V87,N94, T96, K97, R101, L82, P83, L86, V87, N94, T96, K97, F100, R101,L109, M111, L114, V118, L119, A122, G124, G126, T127, Y129, W149, andI194.

In additional embodiments, the variant perhydrolase exhibits a ratio ofperacid hydrolysis of about 0.3 or less, in comparison with wild-typeperhydrolase. In some embodiments, the variant perhydrolase comprises atleast one modification comprising at least one substitution at an aminoacid position equivalent to a position in M. smegmatis perhydrolasecomprising the amino acid sequence set forth in SEQ ID NO:2, wherein atleast one substitution is selected from the group consisting of R4, I5,D10, L12, W14, G15, L12, P18, V19, G22, A23, T25, E26, R27, W34, G36,L38, Q41, L42, G43, A44, I49, E50, E51, G52, L53, S54, A55, R56, T57,N59, T58, D62, T64, D65, R67, L68, N69, G70, S72, Y73, S76, C77, A79,T80, L82, P83, D85, L86, V87, N94, T96, K97, Y99, F100, R101, R102,P104, L109, G110, M111, L114, V118, L119, A122, G124, V125, G126, T127,Y129, W149, F154, and I194.

In yet further embodiments, the variant perhydrolase exhibits a ratio ofperacid hydrolysis of about 0.4 or less, in comparison with wild-typeperhydrolase. In some preferred embodiments, the variant perhydrolasecomprises at least one modification comprising at least one substitutionat an amino acid position equivalent to a position in M. smegmatisperhydrolase comprising the amino acid sequence set forth in SEQ IDNO:2, wherein at least one substitution is selected from the groupconsisting of R4, I5, L6, D10, S11, L12, W14, G15, W16, P18, V19, G22,A23, T25, E26, R27, F28, W34, T35, G36, L38, Q41, L42, G43, A44, D45,E47, I49, E50, E51, G52, L53, S54, A55, R56, T57, T58, N59, T58, I60,D62, T64, D65, R67, L68, N69, G70, S72, Y73, S76, C77, A79, T80, L82,P83, D85, L86, V87, N94, P66, T96, K97, Y99, F100, R101, R102, P104,I107, L109, G110, M111, S112, L114, V118, L119, S121, A122, G124, V125,G126, T127, Y129, W149, F150, F154, I194, and F196.

In some embodiments, the variant perhydrolase exhibits a ratio ofperacid hydrolysis of about 0.5 or less, in comparison with wild-typeperhydrolase. In some preferred embodiments, the variant perhydrolasecomprises at least one modification comprising at least one substitutionat an amino acid position equivalent to a position in M. smegmatisperhydrolase comprising the amino acid sequence set forth in SEQ IDNO:2, wherein at least one substitution is selected from the groupconsisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46,G110, G124, G43, L109, L119, L42, L68, L78, L82, L84, N59, P66, R101,R27, R4, R67, S112, S54, S76, T116, T120, T25, V125, V48, W149, Y73,A44, A79, D85, E51, G124, G126, G15, G52, I194, K97, L119, L12, L38,L53, L68, L86, N94, P18, R101, R27, R4, R67, S54, S72, T58, T80, V118,V87, W34, R4, I5, D10, L12, W14, V19, T25, W34, I49, E50, E51, L53, S54,A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83,L86, V87, N94, T96, F100, R101, L109, M111, L114, L119, W149, Y129,A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, G110, G124, G126,G22, G36, G43, G52, G70, I49, K97, L109, L114, L119, L12, L38, L42, L53,L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, V118, V125, W14,W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150,F196, F28, G110, G124, G36, G43, G52, G70, I107, I5, I60, L109, L119,L53, L6, L68, L82, M111, P104, P66, R102, R67, S11, S112, S121, S54,S72, T25, T35, T57, T58, V118, V125, V19, W149, W16, Y99, G190, V191,G193, T197, N201, D203, L208, A209, V212, L215, and L216.

In additional embodiments, the variant perhydrolase exhibits a ratio ofperacid hydrolysis of about 0.6 or less, in comparison with wild-typeperhydrolase. In some preferred embodiments, the variant perhydrolasecomprises at least one modification comprising at least one substitutionat an amino acid position equivalent to a position in M. smegmatisperhydrolase comprising the amino acid sequence set forth in SEQ IDNO:2, wherein at least one substitution is selected from the groupconsisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46,G110, G124, G43, L109, L119, L42, L68, L78, L82, L84, N59, P66, R101,R27, R4, R67, S112, S54, S76, T116, T120, T25, V125, V48, W149, Y73,A44, A79, D85, E51, G124, G126, G15, G52, I194, K97, L119, L12, L38,L53, L68, L86, N94, P18, R101, R27, R4, R67, S54, S72, T58, T80, V118,V87, W34, R4, I5, D10, L12, W14, V19, T25, W34, I49, E50, E51, L53, S54,A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83,L86, V87, N94, T96, F100, R101, L109, M111, L114, L119, W149, Y129,A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, G110, G124, G126,G22, G36, G43, G52, G70, I49, K97, L109, L114, L119, L12, L38, L42, L53,L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, V118, V125, W14,W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150,F196, F28, G110, G124, G36, G43, G52, G70, I107, I5, I60, L109, L119,L53, L6, L68, L82, M111, P104, P66, R102, R67, S11, S112, S121, S54,S72, T25, T35, T57, T58, V118, V125, V19, W149, W16, A108, A122, A23,A29, A79, C7, D106, D21, D45, D62, D65, D85, E50, F150, F28, G124, G126,G22, G36, G52, I107, I194, K97, L105, L109, L114, L119, L38, L68, L78,L82, L84, M111, N69, N94, P104, P63, P66, R102, R27, S11, S112, S54,S72, T116, T120, T127, T13, T25, T57, T80, T96, V113, V125, V19, W16,Y129, Y73, Y99, G190, V191, G193, T197, N201, D203, L208, A209, V212,L215, and L216.

In yet additional embodiments, the variant perhydrolase exhibits a ratioof peracid hydrolysis of about 0.7 or less, in comparison with wild-typeperhydrolase. In some preferred embodiments, the variant perhydrolasecomprises at least one modification comprising at least one substitutionat an amino acid position equivalent to a position in M. smegmatisperhydrolase comprising the amino acid sequence set forth in SEQ IDNO:2, wherein at least one substitution is selected from the groupconsisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46,G110, G124, G43, L109, L119, L42, L68, L78, L82, L84, N59, P66, R101,R27, R4, R67, S112, S54, S76, T116, T120, T25, V125, V48, W149, Y73,A44, A79, D85, E51, G124, G126, G15, G52, I194, K97, L119, L12, L38,L53, L68, L86, N94, P18, R101, R27, R4, R67, S54, S72, T58, T80, V118,V87, W34, R4, I5, D10, L12, W14, V19, T25, W34, I49, E50, E51, L53, S54,A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83,L86, V87, N94, T96, F100, R101, L109, M111, L114, L119, W149, Y129,A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, G110, G124, G126,G22, G36, G43, G52, G70, I49, K97, L109, L114, L119, L12, L38, L42, L53,L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, V118, V125, W14,W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150,F196, F28, G110, G124, G36, G43, G52, G70, I107, I5, I60, L109, L119,L53, L6, L68, L82, M111, P104, P66, R102, R67, S11, S112, S121, S54,S72, T25, T35, T57, T58, V118, V125, V19, W149, W16, A108, A122, A23,A29, A79, C7, D106, D21, D45, D62, D65, D85, E50, F150, F28, G124, G126,G22, G36, G52, I107, I194, K97, L105, L109, L114, L119, L38, L68, L78,L82, L84, M111, N69, N94, P104, P63, P66, R102, R27, S11, S112, S54,S72, T116, T120, T127, T13, T25, T57, T80, T96, V113, A122, A29, A71,A79, C7, D106, D21, D61, D65, D85, E47, E50, F150, F196, F28, F46, G124,G126, G15, G36, G70, I49, I5, I60, L105, L109, L12, L38, L42, L53, L84,L86, M111, N59, P146, P24, P66, Q41, R102, R27, R56, S112, S121, S54,S72, T116, T120, T127, T128, T13, T57, T64, V125, V17, V19, W14, W149,W16, Y129, Y73, Y99, G190, V191, G193, T197, N201, D203, L208, A209,V212, L215, and L216.

In still further embodiments, the variant perhydrolase exhibits a ratioof peracid hydrolysis of about 0.8 or less, in comparison with wild-typeperhydrolase. In some preferred embodiments, the variant perhydrolasecomprises at least one modification comprising at least one substitutionat an amino acid position equivalent to a position in M. smegmatisperhydrolase comprising the amino acid sequence set forth in SEQ IDNO:2, wherein at least one substitution is selected from the groupconsisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46,G110, G124, G43, L109, L119, L42, L68, L78, L82, L84, N59, P66, R101,R27, R4, R67, S112, S54, S76, T116, T120, T25, V125, V48, W149, Y73,A44, A79, D85, E51, G124, G126, G15, G52, I194, K97, L119, L12, L38,L53, L68, L86, N94, P18, R101, R27, R4, R67, S54, S72, T58, T80, V118,V87, W34, R4, I5, D10, L12, W14, V19, T25, W34, I49, E50, E51, L53, S54,A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83,L86, V87, N94, T96, F100, R101, L109, M111, L114, L119, W149, Y1d29,A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, G110, G124, G126,G22, G36, G43, G52, G70, I49, K97, L109, L114, L119, L12, L38, L42, L53,L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, V118, V125, W14,W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150,F196, F28, G110, G124, G36, G43, G52, G70, I107, I5, I60, L109, L119,L53, L6, L68, L82, M111, P104, P66, R102, R67, S11, S112, S121, S54,S72, T25, T35, T57, T58, V118, V125, V19, W149, W16, A108, A122, A23,A29, A79, C7, D106, D21, D45, D62, D65, D85, E50, F150, F28, G124, G126,G22, G36, G52, I107, I194, K97, L105, L109, L114, L119, L38, L68, L78,L82, L84, M111, N69, N94, P104, P63, P66, R102, R27, S11, S112, S54,S72, T116, T120, T127, T13, T25, T57, T80, T96, V113, A122, A29, A71,A79, C7, D106, D21, D61, D65, D85, E47, E50, F150, F196, F28, F46, G124,G126, G15, G36, G70, I49, I5, I60, L105, L109, L12, L38, L42, L53, L84,L86, M111, N59, P146, P24, P66, Q41, R102, R27, R56, S112, S121, S54,S72, T116, T120, T127, T128, T13, T57, T64, V125, V17, V19, W14, W149,W16, Y129, Y99, A108, A122, A23, A29, A44, A55, A71, A79, C77, D45, D61,D65, D85, D95, E47, E51, F150, F196, F46, G110, G126, G36, G43, G52,I107, I194, I49, I5, I60, I89, L114, L42, L53, L68, L78, L84, M111, N59,N94, P146, P24, P30, P63, P66, P83, Q117, R101, R4, S112, S121, S72,T116, T120, T127, T13, T57, T96, V113, V125, V17, V19, V32, V87, W149,Y129, Y73, G190, V191, G193, T197, N201, D203, L208, A209, V212, L215,and L216.

In additional embodiments, the variant perhydrolase exhibits a ratio ofperacid hydrolysis of about 1.5 or greater, in comparison with wild-typeperhydrolase. In some preferred embodiments, the variant perhydrolasecomprises at least one modification comprising at least one substitutionat an amino acid position equivalent to a position in M. smegmatisperhydrolase comprising the amino acid sequence set forth in SEQ IDNO:2, wherein at least one substitution is selected from the groupconsisting of A122, A23, A29, A55, D45, D62, D65, E26, E50, F150, F46,G110, G124, G43, L109, L119, L42, L68, L78, L82, L84, N59, P66, R101,R27, R4, R67, S112, S54, S76, T116, T120, T25, V125, V48, W149, Y73,A44, A79, D85, E51, G124, G126, G15, G52, I194, K97, L119, L12, L38,L53, L68, L86, N94, P18, R101, R27, R4, R67, S54, S72, T58, T80, V118,V87, W34, R4, I5, D10, L12, W14, V19, T25, W34, I49, E50, E51, L53, S54,A55, R56, N59, D62, T64, D65, R67, L68, N69, S76, C77, T80, L82, P83,L86, V87, N94, T96, F100, R101, L109, M111, L114, L119, W149, Y129,A122, G126, T127, A23, A55, A79, D65, D85, E26, F154, G110, G124, G126,G22, G36, G43, G52, G70, I49, K97, L109, L114, L119, L12, L38, L42, L53,L68, L86, P104, P83, Q41, R102, R56, R67, S54, T57, V118, V125, W14,W149, Y129, Y73, A122, A23, A79, D45, D65, D85, E26, E47, E51, F150,F196, F28, G110, G124, G36, G43, G52, G70, I107, I5, I60, L109, L119,L53, L6, L68, L82, M111, P104, P66, R102, R67, S11, S112, S121, S54,S72, T25, T35, T57, T58, V118, V125, V19, W149, W16, A108, A122, A23,A29, A79, C7, D106, D21, D45, D62, D65, D85, E50, F150, F28, G124, G126,G22, G36, G52, I107, I194, K97, L105, L109, L114, L119, L38, L68, L78,L82, L84, M111, N69, N94, P104, P63, P66, R102, R27, S11, S112, S54,S72, T116, T120, T127, T13, T25, T57, T80, T96, V113, A122, A29, A71,A79, C7, D106, D21, D61, D65, D85, E47, E50, F150, F196, F28, F46, G124,G126, G15, G36, G70, I49, I5, I60, L105, L109, L12, L38, L42, L53, L84,L86, M111, N59, P146, P24, P66, Q41, R102, R27, R56, S112, S121, S54,S72, T116, T120, T127, T128, T13, T57, T64, V125, V17, V19, W14, W149,W16, Y129, Y99, A108, A122, A23, A29, A44, A55, A71, A79, C77, D45, D61,D65, D85, D95, E47, E51, F150, F196, F46, G110, G126, G36, G43, G52,I107, I194, I49, I5, I60, I89, L114, L42, L53, L68, L78, L84, M111, N59,N94, P146, P24, P30, P63, P66, P83, Q117, R101, R4, S112, S121, S72,T116, T120, T127, T13, T57, T96, V113, V125, V17, V19, V32, V87, W149,Y129, and Y73, Y99, A108, A44, C7, D10, D106, D31, D61, D85, E26, E51,F100, F28, F46, G110, G22, G36, G43, G52, G70, I107, I153, I49, I5, I89,K3, L105, L53, L6, L78, L86, M1, N69, P104, P146, P18, P24, P30, P83,Q117, Q40, Q41, R102, R27, R33, R4, S121, S72, S76, T120, T128, T13,T35, T80, T96, V115, V118, V32V48, V87, W34, G190, V191, G193, T197,E198, A199, R202, D203, G205, V206, A209, E210, Q211, S214, and L215.

In additional embodiments, the variant perhydrolase exhibits a ratio ofperacid hydrolysis between about 1.2 and about 1.5, in comparison withwild-type perhydrolase. In some embodiments, the variant perhydrolasecomprises at least one modification comprising at least one substitutionat an amino acid position equivalent to a position in M. smegmatisperhydrolase comprising the amino acid sequence set forth in SEQ IDNO:2, wherein at least one substitution is selected from the groupconsisting of A23, A55, C7, D106, D31, D61, D85, E26, E50, E51, F100,F150, F28, F46, G110, G126, G22, G70, I107, K3, L105, L42, L6, L78,M111, N59, N69, P104, P146, P148, P18, P30, P63, Q117, Q40, Q41, R102,R27, R33, R4, S54, S76, T116, T120, T128, T64, T80, T96, V113, V115,V118, W34, and Y73.

In yet further embodiments, the present invention provides variantperhydrolases in which the variant perhydrolases exhibit a change inperhydrolysis, such that the ratio of variant perhydrolase perhydrolysisto wild-type perhydrolase perhydrolysis is at least about 1.2. In someembodiments, the variant perhydrolase comprises at least onemodification comprising at least one substitution at an amino acidposition equivalent to a position in M. smegmatis perhydrolasecomprising the amino acid sequence set forth in SEQ ID NO:2, wherein atleast one substitution is selected from the group consisting of C7, D10,L12, G15, P18, V19, G22, T25, E26, R27, F28, A29, P30, D31, G36, Q40,Q41, L42, G43, A44, D45, F46, E47, I49, E51, L53, S54, A55, T57, D61,P63, T64, D65, P66, R67, L68, N69, A71, S72, Y73, S76, L78, A79, T80,L82, P83, D85, L86, D95, K97, R101, T103, P104, L105, D106, I107, L109,M111, V113, Q117, V118, S121, G124, V125, G126, T127, P148, F150, I153,F154, and F196.

In further embodiments, the variant perhydrolase exhibits a change inperhydrolysis, such that the ratio of variant perhydrolase perhydrolysisto wild-type perhydrolase perhydrolysis is about 0.8 or less. In someembodiments, the variant perhydrolase comprising at least onemodification comprises at least one substitution at an amino acidposition equivalent to a position in M. smegmatis perhydrolasecomprising the amino acid sequence set forth in SEQ ID NO:2, wherein atleast one substitution is selected from the group consisting of A108,A122, A23, A29, A44, A55, A71, A79, C7, C77, D10, D106, D21, D45, D61,D62, D65, D85, E26, E47, E50, E51, F100, F150, F154, F196, F28, F46,G110, G124, G126, G15, G22, G36, G52, G70, I107, I153, I194, I49, I5,I60, I89, K3, K97, L105, L109, L114, L119, L12, L38, L42, L53, L6, L68,L78, L82, L84, K86, M1, M111, N59N94, P146, P18, P24, P30, P66, P83,Q40, Q41, R101, R102, R27, R33, R4, R56, R67, S11, S112, S54, S72, S76,T103, T116, T120, T127, T128, T13, T25, T35, T57, T64, T80, T96, V113,V115, V118, V125, V17, V19, V32, V48, V87, W13, W149, W16, W34, Y129,Y73, and Y99.

In alternative embodiments, the present invention provides variantperhydrolases comprising at least one modification comprising at leastone substitution at an amino acid position equivalent to a position inM. smegmatis perhydrolase comprising the amino acid sequence set forthin SEQ ID NO:2, wherein at least one substitution is selected from thegroup consisting of A108, A122, A23, A29, A44, A55, A71, A79, C7, C77,D10, D106, D21, D31, D45, D61, D62, D65, D85, E26, E47, E50, E51, F100,F150, F154F196, F28, F46, G110, G124, G126, G15, G22, G36, G43, G52,G70, I107, I153, I194, I49, I5, I60, I89, K3, K97, L105, L109, L114,L119, L12, L38, L42, L53, L6, L68, L78, L82, L84, L86, M1, M111, N59,N69, N94, P104, P146, P148, P18, P24, P30, P63, P66, P83, Q117, Q40,Q41, R101, R102, R27, R33, R4, R56, R67, S11, S112, S121, S54, S72, S76,T103, T116, T120, T127, T128, T13, T25, T35, T57, T58, T64, T80, T96,V113, V115, V118, V125, V17, V19, V32, V48, V87, W14, W149, W16, W34,Y129, Y73, and Y99.

In yet additional embodiments, the variant perhydrolase exhibits achange in perhydrolysis, such that the ratio of variant perhydrolaseperhydrolysis to wild-type perhydrolase perhydrolysis is between about1.2 and about 2. In some embodiments, the variant perhydrolase comprisesat least one modification comprising at least one substitution at anamino acid position equivalent to a position in M. smegmatisperhydrolase comprising the amino acid sequence set forth in SEQ IDNO:2, wherein at least one substitution is selected from the groupconsisting of C7, D10, L12, G15, P18, V19, G22, T25, E26, R27, F28, A29,P30, D31, G36, Q40, Q41, L42, G43, A44, D45, F46, E47, I49, E51, L53,S54, A55, T57, D61, P63, T64, D65, P66, R67, L68, N69, A71, S72, Y73,S76, L78, A79, T80, L82, P83, D85, L86, D95, K97, R101, T103, P104,L105, D106, I107, L109, M111, V113, Q117, V118, S121, G124, V125, G126,T127, P148, F150, I153, F154, F196, G190, E198, A199, R202, D203, V206,A209, E210, Q211, and V212.

In still further embodiments, the variant perhydrolase exhibits a changein perhydrolysis, such that the ratio of variant perhydrolaseperhydrolysis to wild-type perhydrolase perhydrolysis is between about 2and about 2.5. In some embodiments, the variant perhydrolase comprisesat least one modification comprising at least one substitution at anamino acid position equivalent to a position in M. smegmatisperhydrolase comprising the amino acid sequence set forth in SEQ IDNO:2, wherein at least one substitution is selected from the groupconsisting of A44, C7, D10, D85, D95, E26, E47, I107, L12, L42, P104,P148, S54, Q40, Q117, D203, V206, E210.

In still further embodiments, the variant perhydrolase exhibits a changein perhydrolysis, such that the ratio of variant perhydrolaseperhydrolysis to wild-type perhydrolase perhydrolysis is between about2.5 and about 3. In some embodiments, the variant perhydrolase comprisesat least one modification comprising at least one substitution at anamino acid position equivalent to a position in M. smegmatisperhydrolase comprising the amino acid sequence set forth in SEQ IDNO:2, wherein at least one substitution is selected from the groupconsisting of A44, C7, I107, K97, L12, L78, P104, Q40, and V125.

In further embodiments, the variant perhydrolase exhibits a change inperhydrolysis, such that the ratio of variant perhydrolase perhydrolysisto wild-type perhydrolase perhydrolysis is between about 3.0 and about5. In some embodiments, the variant perhydrolase comprises at least onemodification comprising at least one substitution at an amino acidposition equivalent to a position in M. smegmatis perhydrolasecomprising the amino acid sequence set forth in SEQ ID NO:2, wherein atleast one substitution is selected from the group consisting of D10,D85, L53, L78, and S54.

In still further embodiments, the variant perhydrolase exhibits a changein perhydrolysis, such that the ratio of variant perhydrolaseperhydrolysis to wild-type perhydrolase perhydrolysis is about 0.1 orless. In some embodiments, the variant perhydrolase comprises at leastone modification comprising at least one substitution at an amino acidposition equivalent to a position in M. smegmatis perhydrolasecomprising the amino acid sequence set forth in SEQ ID NO:2, wherein atleast one substitution is selected from the group consisting of A23,A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97,L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25,T80, V115, V19, V32, V48, V87, W14, W149, W16, and W34.

In further embodiments, the variant perhydrolase exhibits a change inperhydrolysis, such that the ratio of variant perhydrolase perhydrolysisto wild-type perhydrolase perhydrolysis is about 0.2 or less. In someembodiments, the variant perhydrolase comprises at least onemodification comprising at least one substitution at an amino acidposition equivalent to a position in M. smegmatis perhydrolasecomprising the amino acid sequence set forth in SEQ ID NO:2, wherein atleast one substitution is selected from the group consisting of A23,A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97,L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25,T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62,F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53,L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13,T35, T64, T80, T96, V118, V48, W149, W16, W34, Y129, and Y73.

In additional embodiments, the variant perhydrolase exhibits a change inperhydrolysis, such that the ratio of variant perhydrolase perhydrolysisto wild-type perhydrolase perhydrolysis is about 0.3 or less. In someembodiments, the variant perhydrolase comprises at least onemodification comprising at least one substitution at an amino acidposition equivalent to a position in M. smegmatis perhydrolasecomprising the amino acid sequence set forth in SEQ ID NO:2, wherein atleast one substitution is selected from the group consisting of A23,A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97,L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25,T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62,F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53,L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13,T35, T64, T80, T96, V118, V48, W149, W16, W34, Y129, Y73, A122, A23,A44, C7, D10, D62, F150, G110, G22, G70, I153, I194, I60, I89, K97,L114, L119, L12, L38, L6, L68, L82, M111, N94, P146, Q41, R102, R27, R4,R56, S11, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, andY129.

In yet additional embodiments, the variant perhydrolase exhibits achange in perhydrolysis, such that the ratio of variant perhydrolaseperhydrolysis to wild-type perhydrolase perhydrolysis is about 0.4 orless. In some embodiments, the variant perhydrolase comprises at leastone modification comprising at least one substitution at an amino acidposition equivalent to a position in M. smegmatis perhydrolasecomprising the amino acid sequence set forth in SEQ ID NO:2, wherein atleast one substitution is selected from the group consisting of A23,A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97,L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25,T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62,F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53,L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13,T35, T64, T80, T96, V118, V48, W149, W16, W34, Y129, Y73, A122, A23,A44, C7, D10, D62, F150, G110, G22, G70, I153, I194, I60, I89, K97,L114, L119, L12, L38, L6, L68, L82, M111, N94, P146, Q41, R102, R27, R4,R56, S11, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129,A55, C77, E51, F100, F150, F154, G110, G126, G22, I194, I89, K97, L114,L84, N59, P146, P83, R102, R27, R33, R4, R56, S112, S54, S72, S76, T120,T127, T13, T25, T57, T96, V118, V125, V19, and V87.

In additional embodiments, the variant perhydrolase exhibits a change inperhydrolysis, such that the ratio of variant perhydrolase perhydrolysisto wild-type perhydrolase perhydrolysis is about 0.5 or less. In someembodiments, the variant perhydrolase comprises at least onemodification comprising at least one substitution at an amino acidposition equivalent to a position in M. smegmatis perhydrolasecomprising the amino acid sequence set forth in SEQ ID NO:2, wherein atleast one substitution is selected from the group consisting of A23,A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97,L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25,T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62,F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53,L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13,T35, T64, T80, T96, V118, V48, W149, W16, W34, Y129, Y73, A122, A23,A44, C7, D10, D62, F150, G110, G22, G70, I153, I194, I60, I89, K97,L114, L119, L12, L38, L6, L68, L82, M111, N94, P146, Q41, R102, R27, R4,R56, S11, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129,A55, C77, E51, F100, F150, F154, G110, G126, G22, I194, I89, K97, L114,L84, N59, P146, P83, R102, R27, R33, R4, R56, S112, S54, S72, S76, T120,T127, T13, T25, T57, T96, V118, V125, V19, V87, A23, A55, D10, D23, E26,E50, E51, F150, G110, G126, G15, G36, I107, I49, I5, K97, L109, L119,L12 L38, L6, L68, L84, L86, M111, N59, P146, P24, Q40, R101, R102, R27,R33, R4, R56, S112, S72, S76, T127, T25, T35, T80, T96, V115, V32, V87,W34, and Y129.

In further embodiments, the variant perhydrolase exhibits a change inperhydrolysis, such that the ratio of variant perhydrolase perhydrolysisto wild-type perhydrolase perhydrolysis is about 0.6 or less. In someembodiments, the variant perhydrolase comprises at least onemodification comprising t least one substitution at an amino acidposition equivalent to a position in M. smegmatis perhydrolasecomprising the amino acid sequence set forth in SEQ ID NO:2, wherein atleast one substitution is selected from the group consisting of A23,A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97,L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25,T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62,F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53,L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13,T35, T64, T80, T96, V118, V48, W149, W16, W34, Y129, Y73, A122, A23,A44, C7, D10, D62, F150, G110, G22, G70, I153, I194, I60, I89, K97,L114, L119, L12, L38, L6, L68, L82, M111, N94, P146, Q41, R102, R27, R4,R56, S11, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129,A55, C77, E51, F100, F150, F154, G110, G126, G22, I194, I89, K97, L114,L84, N59, P146, P83, R102, R27, R33, R4, R56, S112, S54, S72, S76, T120,T127, T13, T25, T57, T96, V118, V125, V19, V87, A23, A55, D10, D23, E26,E50, E51, F150, G110, G126, G15, G36, I107, I49, I5, K97, L109, L119,L12 L38, L6, L68, L84, L86, M111, N59, P146, P24, Q40, R101, R102, R27,R33, R4, R56, S112, S72, S76, T127, T25, T35, T80, T96, V115, V32, V87,W34, Y129, A108, A44, A55, D21, D62, F150, g126, G36, G52, I107, I5,I89, L109, L114, L119, L12, L42, L53, L6, L68, L78, L84, P146, P24, P66,P83, R27, S112, S72, S76, T120, T127, T13, T35, T57, T58, T80, T96,V115, V118, V32, V48, V87, W149, and Y73.

In yet further embodiments, the variant perhydrolase exhibits a changein perhydrolysis, such that the ratio of variant perhydrolaseperhydrolysis to wild-type perhydrolase perhydrolysis is about 0.7 orless. In some embodiments, the variant perhydrolase comprises at leastone modification comprising at least one substitution at an amino acidposition equivalent to a position in M. smegmatis perhydrolasecomprising the amino acid sequence set forth in SEQ ID NO:2, wherein atleast one substitution is selected from the group consisting of A23,A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97,L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25,T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62,F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53,L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13,T35, T64, T80, T96, V118, V48, W149, W16, W34, Y129, Y73, A122, A23,A44, C7, D10, D62, F150, G110, G22, G70, I153, I194, I60, I89, K97,L114, L119, L12, L38, L6, L68, L82, M111, N94, P146, Q41, R102, R27, R4,R56, S11, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129,A55, C77, E51, F100, F150, F154, G110, G126, G22, I194, I89, K97, L114,L84, N59, P146, P83, R102, R27, R33, R4, R56, S112, S54, S72, S76, T120,T127, T13, T25, T57, T96, V118, V125, V19, V87, A23, A55, D10, D23, E26,E50, E51, F150, G110, G126, G15, G36, I107, I49, I5, K97, L109, L119,L12 L38, L6, L68, L84, L86, M111, N59, P146, P24, Q40, R101, R102, R27,R33, R4, R56, S112, S72, S76, T127, T25, T35, T80, T96, V115, V32, V87,W34, Y129, A108, A44, A55, D21, D62, F150, g126, G36, G52, I107, I5,I89, L109, L114, L119, L12, L42, L53, L6, L68, L78, L84, P146, P24, P66,P83, R27, S112, S72, S76, T120, T127, T13, T35, T57, T58, T80, T96,V115, V118, V32, V48, V87, W149, Y73, A122, A23, A29, A71, A79, C7, D61,D62, D85, E26, E51, F100, F28, F46, G110, G126, G52, G70, I107, I49, I5,I60, I89, L109, L114, L12, L38, L68, L82, L86, M111, N59, N94, P83,R102, R33, R4, S112, S72, S76, T103, T116, T128, T25, T35, T57, T58,T64, V19, V32, V48, V87, Y129, Y73, and Y99.

In additional embodiments, the variant perhydrolase exhibits a change inperhydrolysis, such that the ratio of variant perhydrolase perhydrolysisto wild-type perhydrolase perhydrolysis is about 0.8 or less. In someembodiments, the variant perhydrolase comprises at least onemodification comprising at least one substitution at an amino acidposition equivalent to a position in M. smegmatis perhydrolasecomprising the amino acid sequence set forth in SEQ ID NO:2, wherein atleast one substitution is selected from the group consisting of A23,A55, D10, D62, F150, F196, F28, G110, G52, G70, I107, I194, I5, K97,L12, L53, L6, L86, N94, P83, R102, R4, R56, S11, S54, T120, T13, T25,T80, V115, V19, V32, V48, V87, W14, W149, W16, W34, A108, A23, A55, D62,F150, F154, G110, G22, G52, G70, I194, K3, K97, L105, L12, L38, L53,L68, L84, N59, N94, P146, P18, R102, R33, R4, R56, S112, S54, T127, T13,T35, T64, T80, T96, V118, V48, W149, W16, W34, Y129, Y73, A122, A23,A44, C7, D10, D62, F150, G110, G22, G70, I153, I194, I60, I89, K97,L114, L119, L12, L38, L6, L68, L82, M111, N94, P146, Q41, R102, R27, R4,R56, S11, S54, T120, T13, T25, T35, T80, V48, W14, W149, W16, W34, Y129,A55, C77, E51, F100, F150, F154, G110, G126, G22, I194, I89, K97, L114,L84, N59, P146, P83, R102, R27, R33, R4, R56, S112, S54, S72, S76, T120,T127, T13, T25, T57, T96, V118, V125, V19, V87, A23, A55, D10, D23, E26,E50, E51, F150, G110, G126, G15, G36, I107, I49, I5, K97, L109, L119,L12, L38, L6, L68, L84, L86, M111, N59, P146, P24, Q40, R101, R102, R27,R33, R4, R56, S112, S72, S76, T127, T25, T35, T80, T96, V115, V32, V87,W34, Y129, A108, A44, A55, D21, D62, F150, g126, G36, G52, I107, I5,I89, L109, L114, L119, L12, L42, L53, L6, L68, L78, L84, P146, P24, P66,P83, R27, S112, S72, S76, T120, T127, T13, T35, T57, T58, T80, T96,V115, V118, V32, V48, V87, W149, Y73, A122, A23, A29, A71, A79, C7, D61,D62, D85, E26, E51, F100, F28, F46, G110, G126, G52, G70, I107, I49, I5,I60, I89, L109, L114, L12, L38, L68, L82, L86, M111, N59, N94, P83,R102, R33, R4, S112, S72, S76, T103, T116, T128, T25, T35, T57, T58,T64, V19, V32, V48, V87, Y129, Y73, Y99, A108, A122, A29, A55, C77, D10,D106, D45, D61, D62, D65, D85, E47, E50, F100, F150, F28, F46, G110,G124, G126, G15, G36, I153, I194, I5, I60, I89, K3, K97, L105, L109,L114, L119, L38, L42, L68, L84, L86, M1, N59, P24, P30, P83, R101, R27,R4, R56, S112, S54, S76, T103, T116, T120, T127, T128, T13, T35, T64,V113, V17, V19, V32, V48, V87, Y129, Y73, and Y99.

The present invention also provides perhydrolase variants, wherein theperhydrolase variants exhibit greater perhydrolysis activity anddecreased peracid hydrolysis activity as compared to wild-typeperhydrolase. In some embodiments, the variant perhydrolases exhibitperhydrolysis activity ratio of at least about 1.2, and peracidhydrolysis activity ratio of about 0.8 or less, as compared to wild-typeperhydrolase. In alternative embodiments, the variant perhydrolasecomprises at least one modification comprising at least one substitutionat an amino acid position equivalent to a position in M. smegmatisperhydrolase comprising the amino acid sequence set forth in SEQ IDNO:2, wherein at least one substitution is selected from the groupconsisting of A29, A44, A55, A71, A79, C7, D10, D106, D31, D85, E26,E47, F150, F154, F196, F28, G124, G126, G36, G43, I153, L109, L42, L53,L109, L42, L53, L109, L42, L53, L68, L82, L86, M111, N69, P104, P148,P18, P63, P66, P83, Q117, Q40, R101, R67, S54, S121, S72, S76, T25, T64,V115, and V19.

In additional embodiments, the perhydrolase exhibits perhydrolysisactivity ratio of at least about 1.2, a peracid hydrolysis activityratio of about 0.8 or less, and a protein concentration ratio of atleast 0.5, as compared to wild-type perhydrolase. In some embodiments,the variant perhydrolase comprises at least one modification comprisingat least one substitution at an amino acid position equivalent to aposition in M. smegmatis perhydrolase comprising the amino acid sequenceset forth in SEQ ID NO:2, wherein at least one substitution is selectedfrom the group consisting of A29, A44, A71, A79, C7, D85, E26, E47, E51,F150, F154, F196, F28, G124, G126, G36, I153, L109, L12, L53, L68, L82,M111, N69, P104, P148, P18, P63, P66, P83, Q117, Q40, R101, R67, S121,S54, S72, S76, T25, T64, V125, and V19.

The present invention provides variant perhydrolases that exhibit anincrease in expression of the perhydrolase variants, as compared to theexpression of wild-type perhydrolase. In some embodiments, the variantperhydrolase comprises at least one modification comprising at least onesubstitution at an amino acid position equivalent to a position in M.smegmatis perhydrolase comprising the amino acid sequence set forth inSEQ ID NO:2, wherein at least one substitution is selected from thegroup consisting of A2, I5, C7, F8, S11, L12, T13, W14, W16, V17, P18,V19, E20, G22, A23, P24, T25, A29, P30, V32, T35, G36, V37, A39, F46,E47, S54, A55, R56, T58, I60, D61, D62, P63, T64, P66, R67, L68, N69,G70, S72, Y73, L74, P75, S76, C77, L78, A79, T80, L82, P83, L84, L86,I89, T93, T96, K97, A98, Y99, F100, R101, R102, T103, P104, L105, D106,I107, A108, L109, G110, S112, V113, L114, V115, T116, Q117, V118, L119,T120, S121, A122, G124, V125, G126, T127, T128, Y129, P130, P132, K133,L135, V136, S138, P141, L142, A143, M145, H147, W149, F150, Q151, I153,G157, Q159, T161, T162, L164, A165, R166, V167, Y168, A170, L171, A172,M175, K176, P178, A182, G183, S184, V185, I186, T188, I194, F196, V191,N201, L208, A209, Q211, Q213, S214, L215, and L216.

The present invention also provides isolated proteins comprisinghomologs of M. smegmatis perhydrolase, wherein the homologs are proteinswithin the SGNH-hydrolase family of proteins. In alternative preferredembodiments, the isolated proteins have at least about 35% identity withthe amino acid sequence of M. smegmatis perhydrolase, in which theprotein comprises at least three residues selected from the groupconsisting of L6, W14, W34, L38, R56, D62, L74, L78, H81, P83, M90, K97,G110, L114, L135, F180, G205, S11, D192, and H195. In furtherembodiments, the perhydrolase is at least approximately about 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous toM. smegmatis perhydrolase. In additional preferred embodiments, theperhydrolase comprises the amino acid sequence set forth in SEQ ID NO:2.

The present invention also provides isolated proteins having at leastabout 38% identity with the amino acid sequence of M. smegmatisperhydrolase, wherein the protein exhibits perhydrolysis activity. Infurther embodiments, the perhydrolase is at least approximately about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%homologous to M. smegmatis perhydrolase. In additional preferredembodiments, the perhydrolase comprises the amino acid sequence setforth in SEQ ID NO:2.

The present invention also provides homologs of M. smegmatisperhydrolase, wherein the homologs are perhydrolases comprising at leastone motif selected from the group consisting of GDSL-GRTT, GDSL-ARTT,GDSN-GRTT, GDSN-ARTT, and SDSL-GRTT. In preferred embodiments, thehomologs exhibit perhydrolysis. In some particularly preferredembodiments, the homologs exhibit a perhydrolysis to hydrolysis ratiothat is great than about 1. In still further embodiments, the homologsare immunologically cross-reactive with antibodies raised against M.smegmatis perhydrolase. In yet additional embodiments, antibodies raisedagainst the homolog cross-react with M. smegmatis perhydrolase.

The present invention also provides isolated proteins having at leastabout 35% identity with the amino acid sequence of at least one M.smegmatis perhydrolase homolog, wherein the proteins exhibitperhydrolysis activity.

In some particularly preferred embodiments, the present inventionprovides proteins having perhydrolase activity, wherein the proteins arein the form of a multimer in solution. In some more preferredembodiments, the protein is a perhydrolase that comprises a dimer. Inalternative particularly preferred embodiments, the protein is aperhydrolase that comprises an octamer. In still further embodiments,the protein is in the form of a multimer in solution and the protein isselected from the group consisting of M. smegmatis perhydrolase, M.smegmatis perhydrolase homologs, and M. smegmatis perhydrolase variants.In yet further embodiments, the protein is selected from the groupconsisting of modified serine hydrolases and modified cysteinehydrolases, wherein the modified serine hydrolases or modified cysteinehydrolases comprise increased perhydrolase activity as compared tounmodified serine hydrolases or unmodified cysteine hydrolases

The present invention also provides proteins having perhydrolaseactivity, wherein the protein comprises at least one motif selected fromthe group consisting of GDSL-GRTT, GDSL-ARTT, GDSN-GRTT, GDSN-ARTT, andSDSL-GRTT. In some embodiments, the protein is obtained from a member ofthe Rhizobiales. In some preferred embodiments, the protein is obtainedfrom a member of the genus Mycobacterium.

The present invention also provides isolated genes identified using atleast one primer selected from the group consisting of SEQ ID NOS:21-69.

The present invention also provides methods for identifying aperhydrolase, comprising the steps of: identifying source of theperhydrolase; analyzing the source to identify sequences comprising atleast one motif selected from the group consisting of GDSL-GRTT,GDSL-ARTT, GDSN-GRTT, GDSN-ARTT, and SDSL-GRTT; expressing the sequencesidentified in step b) to produce the perhydrolase; and testing theperhydrolase for perhydrolysis activity.

In some embodiments, the analyzing step is an amplification step whereinthe primer sequences set forth in SEQ ID NOS:21-69 are used toamplifying the sequences comprising at least one motif selected from thegroup consisting of GDSL-GRTT, GDSL-ARTT, GDSN-GRTT, GDSN-ARTT, andSDSL-GRTT. In still further embodiments, the source is selected from thegroup consisting of environmental sources and metagenomic sources. Thepresent invention also provides proteins identified using the methodsset forth herein. The present invention further provides isolatednucleic acid sequences encoding the proteins identified using themethods set forth herein. In some particularly preferred embodiments,the proteins exhibit a perhydrolysis to hydrolysis ratio that is greaterthan about 1. In still further embodiments, the proteins exhibit aperhydrolysis activity that is at least about 0.2, compared to theperhydrolysis activity exhibited by M. smegmatis perhydrolase. In yetadditional embodiments, the proteins comprise at least three residuesselected from the group consisting of L6, W14, W34, L38, R56, D62, L74,L78, H81, P83, M90, K97, G110, L114, L135, F180, G205, S11, D192, andH195.

In further embodiments, the analyzing step comprises searching at leastone amino acid database. In yet further embodiments, the analyzing stepcomprises searching at least one nucleic acid database to identifynucleic acid sequences encoding the amino acid sequences of theperhydrolase. In still further embodiments, the source is selected fromthe group consisting of environmental sources and metagenomic sources.The present invention further provides isolated nucleic acid sequencesencoding the proteins identified using the methods set forth herein. Insome particularly preferred embodiments, the proteins exhibit aperhydrolysis to hydrolysis ratio that is greater than about 1. In stillfurther embodiments, the proteins exhibit a perhydrolysis activity thatis at least about 0.2, compared to the perhydrolysis activity exhibitedby M. smegmatis perhydrolase. In yet additional embodiments, theproteins comprise at least three residues selected from the groupconsisting of L6, W14, W34, L38, R56, D62, L74, L78, H81, P83, M90, K97,G110, L114, L135, F180, G205, S11, D192, and H195, as set forth in SEQID NO:2.

The present invention also provides variant perhydrolases having alteredsubstrate specificities as compared to wild-type M. smegmatisperhydrolase. In some embodiments, the variant perhydrolases havealtered para nitrophenyl caproate (PNC) activity, as compared towild-type M. smegmatis perhydrolase.

The present invention also provides variant perhydrolases having alteredpI values as compared to wild-type M. smegmatis perhydrolase. In someembodiments, the variant perhydrolases comprise at least one positivelycharged mutation, while in alternative embodiments, the variantperhydrolases comprise at least one negatively charged mutation.

The present invention also provides variant perhydrolases that haveincreased stability, as compared to wild-type M. smegmatis perhydrolase.In some preferred embodiments, the stability of the variant perhydrolaseis selected from the group consisting of thermostability, enzymaticstability, and chemical stability.

The present invention also provides variant perhydrolases, wherein thevariant perhydrolase exhibits at least one altered surface property. Insome preferred embodiments, the variants comprise at least one mutationcomprising at least one substitution at sites selected from the groupconsisting of the residues set forth in Table 15-1.

The present invention also provides perhydrolase variants having atleast one improved property as compared to wild-type perhydrolase.

The present invention also provides expression vectors comprising apolynucleotide sequence encoding at least one perhydrolase variant. Thepresent invention further provides host cells comprising at least onesuch expression vector. In some preferred embodiments, a host cell isselected from the group consisting of Bacillus sp., Streptomyces sp.,Escherichia, and Pantoea sp. The present invention also providesperhydrolases produced by the host cells.

The present invention also provides compositions comprising at least aportion of at least one perhydrolase. In some preferred embodiments, theperhydrolase comprises the amino acid sequence set forth in SEQ ID NO:2.In further embodiments, the perhydrolase is encoded by a polynucleotidesequence comprises SEQ ID NO:1. In additional embodiments, the sequencecomprises at least a portion of SEQ ID NO:1. In further embodiments, thepresent invention provides expression vectors comprising thepolynucleotide sequence encoding at least a portion of at least oneperhydrolase. The present invention also provides host comprising atleast one expression vectors. In some embodiments, the host cells areselected from the group consisting of Bacillus sp., Streptomyces sp.,Escherichia, and Pantoea sp. The present invention also providesperhydrolases produced by these host cells.

The present invention also provides variant perhydrolases, wherein theperhydrolases comprise at least one substitution corresponding to theamino acid positions in SEQ ID NO:2, and wherein the variantperhydrolase has better performance in at least one property, comparedto wild-type M. smegmatis perhydrolase.

The present invention further provides isolated polynucleotidescomprising a nucleotide sequence (i) having at least about 70% identityto SEQ ID NO:1, or (ii) being capable of hybridizing to a probe derivedfrom the nucleotide sequence set forth in SEQ ID NO:1, under conditionsof intermediate to high stringency, or (iii) being complementary to thenucleotide sequence set forth in SEQ ID NO:1. In some embodiments, thepresent invention also provides vectors comprising these polynucleotidesequences. In additional embodiments, the present invention alsoprovides host comprising at least one expression vectors. In someembodiments, the host cells are selected from the group consisting ofBacillus sp., Streptomyces sp., Escherichia, and Pantoea sp. The presentinvention also provides perhydrolases produced by these host cells.

The present invention also provides polynucleotides comprising asequence complementary to at least a portion of the sequence set forthin SEQ ID NO:1.

The present invention also provides methods of producing enzymes havingperhydrolase activity, comprising: transforming a host cell with anexpression vector comprising a polynucleotide having at least 70%sequence identity to SEQ ID NO:1; cultivating the transformed host cellunder conditions suitable for the host cell to produce the perhydrolase;and recovering the perhydrolase. In some preferred embodiments, the hostcell is selected from the group consisting of Streptomyces, Pantoea,Escherichia, and Bacillus species.

The present invention also provides probes comprising a 4 to 150polynucleotide sequence substantially identical to a correspondingfragment of SEQ ID NO:1, wherein the probe is used to detect a nucleicacid sequence coding for an enzyme having perhydrolase activity.

The present invention also provides cleaning compositions comprising: a)at least 0.0001 weight percent of a perhydrolase that exhibits aperhydrolysis to hydrolysis ratio that is greater than 1; b) a moleculecomprising an ester moiety; and c) optionally, an adjunct ingredient.

The present invention further provides cleaning compositions comprising:a) at least 0.0001 weight percent of a perhydrolase that exhibits aperhydrolysis to hydrolysis ratio that is greater than 1; b) a materialselected from the group consisting of a peroxygen source, hydrogenperoxide and mixtures thereof, the peroxygen source being selected fromthe group consisting of: a per-salt; an organic peroxyacid; ureahydrogen peroxide; a carbohydrate and carbohydrate oxidase mixture, andmixtures thereof; c) from about 0.01 to about 50 weight percent of amolecule comprising an ester moiety; and d) optionally, an adjunctingredient.

The present invention also provides cleaning compositions comprising: a)from about 0.0001 to about 1 weight percent of a variant perhydrolasehaving an amino acid sequence comprising at least one modification of anamino acid made at a position equivalent to a position in M. smegmatisperhydrolase comprising the amino acid sequence set forth in SEQ IDNO:2; b) a material selected from the group consisting of a peroxygensource, hydrogen peroxide and mixtures thereof, the peroxygen sourcebeing selected from the group consisting of: a per-salt; an organicperoxyacid; urea hydrogen peroxide; a carbohydrate and carbohydrateoxidase mixture; and mixtures thereof; c) from about 0.01 to about 50weight percent of a molecule comprising an ester moiety; and d)optionally, an adjunct ingredient. In some preferred embodiments, thecleaning compositions further comprise at least one adjunct ingredient.In some particularly preferred embodiments, the adjunct ingredient isselected from the group consisting of surfactants, builders, chelatingagents, dye transfer inhibiting agents, deposition aids, dispersants,enzymes, and enzyme stabilizers, catalytic materials, bleach activators,bleach boosters, preformed peracids, polymeric dispersing agents, claysoil removalanti-redeposition agents, brighteners, suds suppressors,dyes, perfumes, structure elasticizing agents, fabric softeners,carriers, hydrotropes, processing aids, pigments and mixtures thereof.

In additional embodiments, the present invention provides cleaningcompositions wherein: the perhydrolase exhibits a perhydrolysis tohydrolysis molar ratio that is greater than about 0.1; the per-salt isselected from the group consisting of alkalimetal perborate, alkalimetalpercarbonate, alkalimetal perphosphates, alkalimetal persulphates andmixtures thereof; the carbohydrate is selected from the group consistingof mono-carbohydrates, di-carbohydrates, tri-carbohydrates,oligo-carbohydrates and mixtures thereof; the carbohydrate oxidase isselected from the group consisting of aldose oxidase (IUPACclassification EC1.1.3.9), galactose oxidase (IUPAC classificationEC1.1.3.9), cellobiose oxidase (IUPAC classification EC1.1.3.25),pyranose oxidase (IUPAC classification EC1.1.3.10), sorbose oxidase(IUPAC classification EC1.1.3.11) hexose oxidase (IUPAC classificationEC1.1.3.5). glucose oxidase (IUPAC classification EC1.1.3.4) andmixtures thereof; and the molecule comprising an ester moiety has theformula:R¹O_(x)[(R²)_(m)(R³)_(n)]_(p)

(i) wherein R¹ is a moiety selected from the group consisting of H,substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl,alkylaryl, alkylheteroaryl, and heteroaryl;

(ii) each R² is an alkoxylate moiety;

(iii) R³ is an ester-forming moiety having the formula:

R⁴CO— wherein R⁴ is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl,alkylheteroaryl, and heteroaryl;

(iv) x is 1 when R¹ is H; when R¹ is not H, x is an integer that isequal to or less than the number of carbons in R¹;

(v) p is an integer that is equal to or less than x;

(vi) m is an integer from 0 to 50; and

(vii) n is at least 1

In alternative embodiments, the present invention provides cleaningcompositions wherein: a) R¹ is an C₂-C₃₂ substituted or unsubstitutedalkyl or heteroalkyl moiety; b) each R² is independently an ethoxylateor propoxylate moiety; and c) m is an integer from 1 to 12. In someembodiments, R³ is an ester-forming moiety having the formula: R⁴CO—wherein R⁴ is: a) a substituted or unsubstituted alkyl, alkenyl oralkynyl moiety comprising from 1 to 22 carbon atoms; or b) a substitutedor unsubstituted aryl, alkylaryl, alkylheteroaryl or heteroaryl moietycomprising from 4 to 22 carbon atoms.

In still further embodiments of the cleaning compositions, the moleculecomprising the ester moiety has the formula:R¹O_(x)[(R²)_(m)(R³)_(n)]_(p)

wherein: a) R¹ is H or a moiety that comprises a primary, secondary,tertiary or quaternary amine moiety, the R¹ moiety that comprises anamine moiety being selected from the group consisting of substituted orunsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl,alkylheteroaryl, and heteroaryl; b) each R² is an alkoxylate moiety; c)R³ is an ester-forming moiety having the formula: R⁴CO— wherein R⁴ maybe H, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl,alkylaryl, alkylheteroaryl, and heteroaryl; d) x is 1 when R¹ is H; whenR¹ is not H, x is an integer that is equal to or less than the number ofcarbons in R¹; e) p is an integer that is equal to or less than x; f) mis an integer from 0 to 12; and g) n is at least 1.

In still further embodiments of the present cleaning compositions, themolecule comprising an ester moiety has a weight average molecularweight of less than 600,000 Daltons. In yet additional embodiments, anadjunct ingredient is selected from the group consisting of surfactants,builders, chelating agents, dye transfer inhibiting agents, depositionaids, dispersants, enzymes, and enzyme stabilizers, catalytic materials,bleach activators, bleach boosters, preformed peracids, polymericdispersing agents, clay soil removal/anti-redeposition agents,brighteners, suds suppressors, dyes, perfumes, structure elasticizingagents, fabric softeners, carriers, hydrotropes, processing aids,pigments and mixtures thereof.

The present invention further provides methods of cleaning comprisingthe steps of: a) contacting a surface and/or an article comprising afabric with any of the cleaning compositions provided above and/or acomposition comprising any of the cleaning compositions provided above;and b) optionally washing and/or rinsing the surface or material.

In alternative embodiments, the present invention provides methods ofcleaning, the method comprising the steps of: a) contacting a surfaceand/or an article comprising a fabric with any suitable cleaningcomposition provided above and/or a composition comprising any suitablecleaning provided above; and b) optionally washing and/or rinsing thesurface or material.

The present invention also provides bleaching compositions comprising atleast one perhydrolase. In some particularly preferred embodiments, theperhydrolase exhibits a perhydrolysis to hydrolysis ratio that isgreater than 1. In some embodiments, the bleaching compositions furthercomprise at least one additional enzymes or enzyme derivatives selectedfrom the group consisting of proteases, amylases, lipases, mannanases,pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides bleaching compositions comprising atleast one perhydrolase variant having an amino acid sequence comprisingat least one modification of an amino acid made at a position equivalentto a position in M. smegmatis perhydrolase comprising the amino acidsequence set forth in SEQ ID NO:2. In some particularly preferredembodiments, the perhydrolase exhibits a perhydrolysis to hydrolysisratio that is greater than 1. In some embodiments, the bleachingcompositions further comprise at least one additional enzymes or enzymederivatives selected from the group consisting of proteases, amylases,lipases, mannanases, pectinases, cutinases, oxidoreductases,hemicellulases, and cellulases.

The present invention also provides bleaching compositions comprising atleast one perhydrolase variant having at least one improved property ascompared to wild-type perhydrolase. In some particularly preferredembodiments, the perhydrolase exhibits a perhydrolysis to hydrolysisratio that is greater than 1. In some embodiments, the bleachingcompositions further comprise at least one additional enzymes or enzymederivatives selected from the group consisting of proteases, amylases,lipases, mannanases, pectinases, cutinases, oxidoreductases,hemicellulases, and cellulases.

The present invention also provides bleaching compositions comprising atleast one perhydrolase variant comprising at least one substitutioncorresponding to the amino acid positions in SEQ ID NO:2, and whereinthe variant perhydrolase has better performance in at least one propertycompared to wild-type M. smegmatis perhydrolase. In some particularlypreferred embodiments, the perhydrolase exhibits a perhydrolysis tohydrolysis ratio that is greater than 1. In some embodiments, thebleaching compositions further comprise at least one additional enzymesor enzyme derivatives selected from the group consisting of proteases,amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases,hemicellulases, and cellulases.

The present invention also provides bleaching compositions comprising atleast one perhydrolase that is at least approximately about 35%homologous to M. smegmatis perhydrolase. In some particularly preferredembodiments, the perhydrolase exhibits a perhydrolysis to hydrolysisratio that is greater than 1. In some embodiments, the bleachingcompositions further comprise at least one additional enzymes or enzymederivatives selected from the group consisting of proteases, amylases,lipases, mannanases, pectinases, cutinases, oxidoreductases,hemicellulases, and cellulases.

The present invention also provides disinfecting compositions comprisingat least one perhydrolase. In some particularly preferred embodiments,the perhydrolase exhibits a perhydrolysis to hydrolysis ratio that isgreater than 1. In some embodiments, the bleaching compositions furthercomprise at least one additional enzymes or enzyme derivatives selectedfrom the group consisting of proteases, amylases, lipases, mannanases,pectinases, cutinases, oxidoreductases, hemicellulases, and cellulases.

The present invention also provides disinfecting compositions comprisingat least one perhydrolase variant having an amino acid sequencecomprising at least one modification of an amino acid made at a positionequivalent to a position in M. smegmatis perhydrolase comprising theamino acid sequence set forth in SEQ ID NO:2. In some particularlypreferred embodiments, the perhydrolase exhibits a perhydrolysis tohydrolysis ratio that is greater than 1. In some embodiments, thebleaching compositions further comprise at least one additional enzymesor enzyme derivatives selected from the group consisting of proteases,amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases,hemicellulases, and cellulases.

The present invention also provides disinfecting compositions comprisingat least one perhydrolase variant having at least one improved propertyas compared to wild-type perhydrolase. In some particularly preferredembodiments, the perhydrolase exhibits a perhydrolysis to hydrolysisratio that is greater than 1. In some embodiments, the bleachingcompositions further comprise at least one additional enzymes or enzymederivatives selected from the group consisting of proteases, amylases,lipases, mannanases, pectinases, cutinases, oxidoreductases,hemicellulases, and cellulases.

The present invention also provides disinfecting compositions comprisingat least one perhydrolase variant comprising at least one substitutioncorresponding to the amino acid positions in SEQ ID NO:2, and whereinthe variant perhydrolase has better performance in at least one propertycompared to wild-type M. smegmatis perhydrolase. In some particularlypreferred embodiments, the perhydrolase exhibits a perhydrolysis tohydrolysis ratio that is greater than 1. In some embodiments, thebleaching compositions further comprise at least one additional enzymesor enzyme derivatives selected from the group consisting of proteases,amylases, lipases, mannanases, pectinases, cutinases, oxidoreductases,hemicellulases, and cellulases.

The present invention also provides disinfecting compositions comprisingat least one perhydrolase that is at least approximately about 35%homologous to M. smegmatis perhydrolase. In some particularly preferredembodiments, the perhydrolase exhibits a perhydrolysis to hydrolysisratio that is greater than 1. In some embodiments, the bleachingcompositions further comprise at least one additional enzymes or enzymederivatives selected from the group consisting of proteases, amylases,lipases, mannanases, pectinases, cutinases, oxidoreductases,hemicellulases, and cellulases.

In some preferred embodiments, the perhydrolase is at leastapproximately 70% homologous to M. smegmatis perhydrolase comprising theamino acid sequence set forth in SEQ ID NO:2. In some embodiments, thepresent invention provides perhydrolases that cross react with antibodygenerated against M. smegmatis perhydrolase, particularly thatcomprising the amino acid sequence set forth in SEQ ID NO:2. In furtherembodiments, the present invention provides perhydrolases that arestructural homologs of the M. smegmatis perhydrolase, in which activesite comprises sites homologous to S11, D192, and H195 of the M.smegmatis perhydrolase. In yet additional embodiments, the presentinvention provides perhydrolases comprising one or more modifications atthe following residues: Cys7, Asp10, Ser11, Leu12, Thr13, Trp14, Trp16,Pro24, Thr25, Leu53, Ser54, Ala55, Thr64, Asp65, Arg67, Cys77, Thr91,Asn94, Asp95, Tyr99, Val125, Pro138, Leu140, Pro146, Pro148, Trp149,Phe150, Ile153, Phe154, Thr159, Thr186, Ile192, Ile194, and Phe196.However, it is not intended that the present invention be limited toperhydrolases with these modifications only at these residues, asperhydrolases with other modifications also find use with the presentinvention.

In some embodiments, at least one perhydrolase of the present inventionis used in a cleaning process wherein an article to be cleaned isexposed to a sufficient amount of the at least one perhydrolase underconditions such that the perhydrolase cleans and/or bleaches, and/ordecolorizes any/all stains present on the article (e.g., laundry anddish detergents). In some embodiments, the cleaning further comprisesdisinfecting. In some embodiments, the article cleaned, bleached and/ordisinfected using at least one perhydrolase of the present inventioncomprises textiles and/or hard surfaces, while in other embodiments, thearticle is paper or pulp, and in still further embodiments, at least oneperhydrolase is used as a personal care product to whiten or bleachhair, teeth, skin, etc. Thus, in some embodiments, the present inventionprovides compositions for use in various cleaning, bleaching, and/ordisinfecting applications. Indeed, it is not intended that the presentinvention be limited to any particular application.

In some preferred embodiments, the perhydrolase comprises SEQ ID NO:2.In some preferred alternative embodiments, the perhydrolase is encodedby the nucleic acid sequence set forth in SEQ ID NO:1.

In some embodiments, the present invention provides enzymes withactivities that result in high peracid/acid ratios. In alternativeembodiments, the present invention provides the perhydrolase ofMycobacterium smegmatis, as well as sequence and/or structural homologsof this protein. In additional embodiments, the present inventionprovides enzymes that have been modified so as to express perhydrolaseactivity with a high perhydrolysis to hydrolase ratio either in additionto or instead of the enzyme's original activity. In additionalembodiments, the present invention provides modified enzymes withaltered substrate specificity, Km, kcat, perhydrolase activity, and/orperacid degradation activity.

In additional embodiments, the present invention provides means toidentify, produce, and characterize enzymes that comprise theperhydrolysis activity of the present invention. The present inventionfurther provides methods and compositions comprising at least oneperhydrolase for cleaning, disinfecting, bleaching, and otherapplications, including but not limited to paper and pulp bleaching,fabric and garment cleaning, hard surface cleaning, and personal careapplications (e.g., oral care, hair care, and skin care). In somepreferred embodiments, the present invention provides methods andcompositions for bleaching cotton and other fabrics. Indeed, the presentinvention finds use in the bleaching and cleaning of various textiles.It is not intended that the present invention be limited to anyparticular setting, application or use, as it is contemplated that itwill find use in numerous areas where an enzymatic generation ofperacids is desired over the use of preformed peracids or hydrogenperoxide or other bleaching chemicals, under conditions including butnot limited to a wide range of pHs and temperatures. The presentinvention also finds use in applications where peracid hydrolysis isuseful, such as in the clean up of peracids.

Furthermore, the present invention provides means to produceperhydrolase enzymes suitable for cleaning, disinfecting, bleaching, andother applications, including personal care.

DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B provide a phylogenetic tree of M. smegmatis perhydrolaseand other related sequences.

FIG. 2 provides an overview phylogenetic tree, showing the majorbranches of the bacteria and the origin of the active clones/sequencescompared to M. smegmatis.

FIG. 3 provides a schematic of four structural families of serinehydrolases, including perhydrolase (SGNH-hydrolase family),chymotrypsin, subtilisin, and α/β hydrolase.

FIG. 4 provides a diagram of the structure of the perhydrolase fold.

FIG. 5 provides a map of plasmid pET26-M4aE11.

FIG. 6 provides a purification table showing the enzyme activity of theenzyme of the present invention through various steps in thepurification process.

FIG. 7 provides a graph which shows the ratio of perbutyric acid tobutyric acid generated by various enzymes from 10 mM tributyrin and 29mM hydrogen peroxide in 40 minutes.

FIG. 8 provides a graph showing the peracid production by 30 mM acetateequivalents and 29 mM hydrogen peroxide, tested at various pHs. Theseresults show that using the perhydrolase composition of the presentinvention, there is peracid generation over a wide pH range. Incontrast, with TAED and hydrogen peroxide, peracid generation is limitedto alkaline conditions.

FIG. 9 provides a graph showing the peracid production by 0.1 ppmperhydrolase enzyme in 30 mM ethyl acetate and 20 mM hydrogen peroxideat various temperatures. These results show that the perhydrolase of thepresent invention works at a wide range of temperatures, including lowtemperatures.

FIG. 10 provides a graph showing the ratio of perbutyric acid to butyricacid generated by various enzymes from 10 mM tributyrin and 29 mMhydrogen peroxide in 4, 10, and 30 minutes.

FIG. 11 provides a graph showing the ratio of peracetic acid to aceticacid generated by various enzymes from 10 mM triacetin and 29 mMhydrogen peroxide in 4 and 10 minutes.

FIG. 12 provides a map of plasmid pMSATNcoI.

FIG. 13 provides a map of plasmid pMSATNco1-1.

FIG. 14 provides a map of plasmid pAH505.

FIG. 15 provides a map of plasmid pSFNASally.

FIG. 16 provides a map of plasmid pCP606.

FIG. 17 provides a map of plasmid pCP649.

FIG. 18 provides a map of plasmid pSECGT-MSAT.

FIG. 19 provides a map of plasmid pSEGT-phdA4.

FIG. 20 provides a map of plasmid pMC355rbs.

FIG. 21 provides a graph showing the degree of bleaching by threedetergents tested alone and in comparison with the M. smegmatisperhydrolase of the present invention.

FIG. 22 provides a graph showing the bleaching ability of the M.smegmatis perhydrolase tested on cotton.

FIG. 23 provides a graph showing the bleaching ability of the M.smegmatis perhydrolase tested on linen.

DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions comprising atleast one perhydrolase enzyme for cleaning and other applications. Insome particularly preferred embodiments, the present invention providesmethods and compositions for generation of peracids. In particular, thepresent invention provides improved methods and compositions comprisingperhydrolysis enzymes with high peracid/acid ratios for cleaning,bleaching, disinfecting and other applications. In some preferredembodiments, the present invention provides improved methods andcompositions for generation of peracids. The present invention findsparticular use in applications involving cleaning, bleaching anddisinfecting.

Unless otherwise indicated, the practice of the present inventioninvolves conventional techniques commonly used in molecular biology,microbiology, protein purification, protein engineering, protein and DNAsequencing, and recombinant DNA fields, which are within the skill ofthe art. Such techniques are known to those of skill in the art and aredescribed in numerous texts and reference works (See e.g., Sambrook etal., “Molecular Cloning: A Laboratory Manual”, Second Edition (ColdSpring Harbor), [1989]); and Ausubel et al., “Current Protocols inMolecular Biology” [1987]). All patents, patent applications, articlesand publications mentioned herein, both supra and infra, are herebyexpressly incorporated herein by reference.

Furthermore, the headings provided herein are not limitations of thevarious aspects or embodiments of the invention which can be had byreference to the specification as a whole. Accordingly, the termsdefined immediately below are more fully defined by reference to thespecification as a whole. Nonetheless, in order to facilitateunderstanding of the invention, a number of terms are defined below.

DEFINITIONS

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. For example,Singleton and Sainsbury, Dictionary of Microbiology and MolecularBiology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham,The Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991)provide those of skill in the art with a general dictionaries of many ofthe terms used in the invention. Although any methods and materialssimilar or equivalent to those described herein find use in the practiceof the present invention, the preferred methods and materials aredescribed herein. Accordingly, the terms defined immediately below aremore fully described by reference to the Specification as a whole. Also,as used herein, the singular terms “a”, “an,” and “the” include theplural reference unless the context clearly indicates otherwise. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively. It is to be understood that thisinvention is not limited to the particular methodology, protocols, andreagents described, as these may vary, depending upon the context theyare used by those of skill in the art.

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

As used herein, the term “bleaching” refers to the treatment of amaterial (e.g., fabric, laundry, pulp, etc.) or surface for a sufficientlength of time and under appropriate pH and temperature conditions toeffect a brightening (i.e., whitening) and/or cleaning of the material.Examples of chemicals suitable for bleaching include but are not limitedto ClO₂, H₂O₂, peracids, NO₂, etc.

As used herein, the term “disinfecting” refers to the removal ofcontaminants from the surfaces, as well as the inhibition or killing ofmicrobes on the surfaces of items. It is not intended that the presentinvention be limited to any particular surface, item, or contaminant(s)or microbes to be removed.

As used herein, the term “perhydrolase” refers to an enzyme that iscapable of catalyzing a reaction that results in the formation ofsufficiently high amounts of peracid suitable for applications such ascleaning, bleaching, and disinfecting. In particularly preferredembodiments, the perhydrolase enzymes of the present invention producevery high perhydrolysis to hydrolysis ratios. The high perhydrolysis tohydrolysis ratios of these distinct enzymes makes these enzymes suitablefor use in a very wide variety of applications. In additional preferredembodiments, the perhydrolases of the present invention arecharacterized by having distinct tertiary structure and primarysequence. In particularly preferred embodiments, the perhydrolases ofthe present invention comprises distinct primary and tertiarystructures. In some particularly preferred embodiments, theperhydrolases of the present invention comprise distinct quaternarystructure. In some preferred embodiments, the perhydrolase of thepresent invention is the M. smegmatis perhydrolase, while in alternativeembodiments, the perhydrolase is a variant of this perhydrolase, whilein still further embodiments, the perhydrolase is a homolog of thisperhydrolase. In further preferred embodiments, a monomeric hydrolase isengineered to produce a multimeric enzyme that has better perhydrolaseactivity than the monomer. However, it is not intended that the presentinvention be limited to this specific M. smegmatis perhydrolase,specific variants of this perhydrolase, nor specific homologs of thisperhydrolase.

As used herein, the term “multimer” refers to two or more proteins orpeptides that are covalently or non-covalently associated and exist as acomplex in solution. A “dimer” is a multimer that contains two proteinsor peptides; a “trimer” contains three proteins or peptides, etc. Asused herein, “octamer” refers to a multimer of eight proteins orpeptides.

As used herein, the phrase “perhydrolysis to hydrolysis ratio” is theratio of the amount of enzymatically produced peracid to that ofenzymatically produced acid by the perhydrolase, under definedconditions and within a defined time. In some preferred embodiments, theassays provided herein are used to determine the amounts of peracid andacid produced by the enzyme.

As used herein, “personal care products” means products used in thecleaning, bleaching and/or disinfecting of hair, skin, scalp, and teeth,including, but not limited to shampoos, body lotions, shower gels,topical moisturizers, toothpaste, and/or other topical cleansers. Insome particularly preferred embodiments, these products are utilized onhumans, while in other embodiments, these products find use withnon-human animals (e.g., in veterinary applications).

As used herein, “pharmaceutically-acceptable” means that drugs,medicaments and/or inert ingredients which the term describes aresuitable for use in contact with the tissues of humans and other animalswithout undue toxicity, incompatibility, instability, irritation,allergic response, and the like, commensurate with a reasonablebenefit/risk ratio.

As used herein, “cleaning compositions” and “cleaning formulations”refer to compositions that find use in the removal of undesiredcompounds from items to be cleaned, such as fabric, dishes, contactlenses, other solid substrates, hair (shampoos), skin (soaps andcreams), teeth (mouthwashes, toothpastes) etc. The term encompasses anymaterials/compounds selected for the particular type of cleaningcomposition desired and the form of the product (e.g., liquid, gel,granule, or spray composition), as long as the composition is compatiblewith the perhydrolase and other enzyme(s) used in the composition. Thespecific selection of cleaning composition materials are readily made byconsidering the surface, item or fabric to be cleaned, and the desiredform of the composition for the cleaning conditions during use.

The terms further refer to any composition that is suited for cleaning,bleaching, disinfecting, and/or sterilizing any object and/or surface.It is intended that the terms include, but are not limited to detergentcompositions (e.g., liquid and/or solid laundry detergents and finefabric detergents; hard surface cleaning formulations, such as forglass, wood, ceramic and metal counter tops and windows; carpetcleaners; oven cleaners; fabric fresheners; fabric softeners; andtextile and laundry pre-spotters, as well as dish detergents).

Indeed, the term “cleaning composition” as used herein, includes unlessotherwise indicated, granular or powder-form all-purpose or heavy-dutywashing agents, especially cleaning detergents; liquid, gel orpaste-form all-purpose washing agents, especially the so-calledheavy-duty liquid (HDL) types; liquid fine-fabric detergents; handdishwashing agents or light duty dishwashing agents, especially those ofthe high-foaming type; machine dishwashing agents, including the varioustablet, granular, liquid and rinse-aid types for household andinstitutional use; liquid cleaning and disinfecting agents, includingantibacterial hand-wash types, cleaning bars, mouthwashes, denturecleaners, car or carpet shampoos, bathroom cleaners; hair shampoos andhair-rinses; shower gels and foam baths and metal cleaners; as well ascleaning auxiliaries such as bleach additives and “stain-stick” orpre-treat types.

As used herein, the terms “detergent composition” and “detergentformulation” are used in reference to mixtures which are intended foruse in a wash medium for the cleaning of soiled objects. In somepreferred embodiments, the term is used in reference to launderingfabrics and/or garments (e.g., “laundry detergents”). In alternativeembodiments, the term refers to other detergents, such as those used toclean dishes, cutlery, etc. (e.g., “dishwashing detergents”). It is notintended that the present invention be limited to any particulardetergent formulation or composition. Indeed, it is intended that inaddition to perhydrolase, the term encompasses detergents that containsurfactants, transferase(s), hydrolytic enzymes, oxido reductases,builders, bleaching agents, bleach activators, bluing agents andfluorescent dyes, caking inhibitors, masking agents, enzyme activators,antioxidants, and solubilizers.

As used herein, “enhanced performance” in a detergent is defined asincreasing cleaning of bleach-sensitive stains (e.g., grass, tea, wine,blood, dingy, etc.), as determined by usual evaluation after a standardwash cycle. In particular embodiments, the perhydrolase of the presentinvention provides enhanced performance in the oxidation and removal ofcolored stains and soils. In further embodiments, the perhydrolase ofthe present invention provides enhanced performance in the removaland/or decolorization of stains. In yet additional embodiments, theperhydrolase of the present invention provides enhanced performance inthe removal of lipid-based stains and soils. In still furtherembodiments, the perhydrolase of the present invention provides enhancedperformance in removing soils and stains from dishes and other items.

As used herein the term “hard surface cleaning composition,” refers todetergent compositions for cleaning hard surfaces such as floors, walls,tile, bath and kitchen fixtures, and the like. Such compositions areprovided in any form, including but not limited to solids, liquids,emulsions, etc.

As used herein, “dishwashing composition” refers to all forms forcompositions for cleaning dishes, including but not limited to granularand liquid forms.

As used herein, “fabric cleaning composition” refers to all forms ofdetergent compositions for cleaning fabrics, including but not limitedto, granular, liquid and bar forms.

As used herein, “textile” refers to woven fabrics, as well as staplefibers and filaments suitable for conversion to or use as yarns, woven,knit, and non-woven fabrics. The term encompasses yarns made fromnatural, as well as synthetic (e.g., manufactured) fibers.

As used herein, “textile materials” is a general term for fibers, yarnintermediates, yarn, fabrics, and products made from fabrics (e.g.,garments and other articles).

As used herein, “fabric” encompasses any textile material. Thus, it isintended that the term encompass garments, as well as fabrics, yarns,fibers, non-woven materials, natural materials, synthetic materials, andany other textile material.

As used herein, the term “compatible,” means that the cleaningcomposition materials do not reduce the enzymatic activity of theperhydrolase to such an extent that the perhydrolase is not effective asdesired during normal use situations. Specific cleaning compositionmaterials are exemplified in detail hereinafter.

As used herein, “effective amount of perhydrolase enzyme” refers to thequantity of perhydrolase enzyme necessary to achieve the enzymaticactivity required in the specific application (e.g., personal careproduct, cleaning composition, etc.). Such effective amounts are readilyascertained by one of ordinary skill in the art and are based on manyfactors, such as the particular enzyme variant used, the cleaningapplication, the specific composition of the cleaning composition, andwhether a liquid or dry (e.g., granular, bar) composition is required,and the like.

As used herein, “non-fabric cleaning compositions” encompass hardsurface cleaning compositions, dishwashing compositions, personal carecleaning compositions (e.g., oral cleaning compositions, denturecleaning compositions, personal cleansing compositions, etc.), andcompositions suitable for use in the pulp and paper industry.

As used herein, “oral cleaning compositions” refers to dentifrices,toothpastes, toothgels, toothpowders, mouthwashes, mouth sprays, mouthgels, chewing gums, lozenges, sachets, tablets, biogels, prophylaxispastes, dental treatment solutions, and the like. Oral care compositionsthat find use in conjunction with the perhydrolases of the presentinvention are well known in the art (See e.g., U.S. Pat. Nos. 5,601,750,6,379,653, and 5,989,526, all of which are incorporated herein byreference).

As used herein, “pulp treatment compositions” refers to the use of thepresent perhydrolase enzymes in compositions suitable for use inpapermaking. It is intended that the term encompass compositionssuitable for the treatment of any pulp material, including wood, as wellas non-wood materials, such as “agricultural residues” and “fibercrops,” including but not limited to wheat straw, rice straw, cornstalks, bagasse (sugar cane), rye grass straw, seed flax straw, flaxstraw, kenaf, industrial hemp, sisal, textile flat straw, hesperaloe,etc. Thus, the present invention also encompasses the use of theperhydrolases of the present invention in pulp treatment methods.

As used herein, “oxidizing chemical” refers to a chemical that has thecapability of bleaching pulp or any other material. The oxidizingchemical is present at an amount, pH and temperature suitable forbleaching. The term includes, but is not limited to hydrogen peroxideand peracids.

As used herein, “acyl” is the general name for organic acid groups,which are the residues of carboxylic acids after removal of the —OHgroup (e.g., ethanoyl chloride, CH₃CO—Cl, is the acyl chloride formedfrom ethanoic acid, CH₃COO—H). The names of the individual acyl groupsare formed by replacing the “-ic” of the acid by “-yl.”

As used herein, the term “acylation” refers to the chemicaltransformation which substitutes the acyl (RCO—) group into a molecule,generally for an active hydrogen of an —OH group.

As used herein, the term “transferase” refers to an enzyme thatcatalyzes the transfer of functional compounds to a range of substrates.

As used herein, “leaving group” refers to the nucleophile which iscleaved from the acyl donor upon substitution by another nucleophile.

As used herein, the term “enzymatic conversion” refers to themodification of a substrate to an intermediate or the modification of anintermediate to an end-product by contacting the substrate orintermediate with an enzyme. In some embodiments, contact is made bydirectly exposing the substrate or intermediate to the appropriateenzyme. In other embodiments, contacting comprises exposing thesubstrate or intermediate to an organism that expresses and/or excretesthe enzyme, and/or metabolizes the desired substrate and/or intermediateto the desired intermediate and/or end-product, respectively.

As used herein, the phrase “detergent stability” refers to the stabilityof a detergent composition. In some embodiments, the stability isassessed during the use of the detergent, while in other embodiments,the term refers to the stability of a detergent composition duringstorage.

As used herein, the phrase, “stability to proteolysis” refers to theability of a protein (e.g., an enzyme) to withstand proteolysis. It isnot intended that the term be limited to the use of any particularprotease to assess the stability of a protein.

As used herein, “oxidative stability” refers to the ability of a proteinto function under oxidative conditions. In particular, the term refersto the ability of a protein to function in the presence of variousconcentrations of H₂O₂ and/or peracid. Stability under various oxidativeconditions can be measured either by standard procedures known to thosein the art and/or by the methods described herein. A substantial changein oxidative stability is evidenced by at least about a 5% or greaterincrease or decrease (in most embodiments, it is preferably an increase)in the half-life of the enzymatic activity, as compared to the enzymaticactivity present in the absence of oxidative compounds.

As used herein, “pH stability” refers to the ability of a protein tofunction at a particular pH. In general, most enzymes have a finite pHrange at which they will function. In addition to enzymes that functionin mid-range pHs (i.e., around pH 7), there are enzymes that are capableof working under conditions with very high or very low pHs. Stability atvarious pHs can be measured either by standard procedures known to thosein the art and/or by the methods described herein. A substantial changein pH stability is evidenced by at least about 5% or greater increase ordecrease (in most embodiments, it is preferably an increase) in thehalf-life of the enzymatic activity, as compared to the enzymaticactivity at the enzyme's optimum pH. However, it is not intended thatthe present invention be limited to any pH stability level nor pH range.

As used herein, “thermal stability” refers to the ability of a proteinto function at a particular temperature. In general, most enzymes have afinite range of temperatures at which they will function. In addition toenzymes that work in mid-range temperatures (e.g., room temperature),there are enzymes that are capable of working in very high or very lowtemperatures. Thermal stability can be measured either by knownprocedures or by the methods described herein. A substantial change inthermal stability is evidenced by at least about 5% or greater increaseor decrease (in most embodiments, it is preferably an increase) in thehalf-life of the catalytic activity of a mutant when exposed to adifferent temperature (i.e., higher or lower) than optimum temperaturefor enzymatic activity. However, it is not intended that the presentinvention be limited to any temperature stability level nor temperaturerange.

As used herein, the term “chemical stability” refers to the stability ofa protein (e.g., an enzyme) towards chemicals that adversely affect itsactivity. In some embodiments, such chemicals include, but are notlimited to hydrogen peroxide, peracids, anionic detergents, cationicdetergents, non-ionic detergents, chelants, etc. However, it is notintended that the present invention be limited to any particularchemical stability level nor range of chemical stability.

As used herein, the phrase “perhydrolase activity improvement” refers tothe relative improvement of perhydrolase activity, in comparison with astandard enzyme. In some embodiments, the term refers to an improvedrate of perhydrolysis product, while in other embodiments, the termencompasses perhydrolase compositions that produce less hydrolysisproduct. In additional embodiments, the term refers to perhydrolasecompositions with altered substrate specificity.

As used herein, the phrase “alteration in substrate specificity” refersto changes in the substrate specificity of an enzyme. In someembodiments, a change in substrate specificity is defined as adifference between the K_(cat)/K_(m) ratio observed with an enzymecompared to enzyme variants or other enzyme compositions. Enzymesubstrate specificities vary, depending upon the substrate tested. Thesubstrate specificity of an enzyme is determined by comparing thecatalytic efficiencies it exhibits with different substrates. Thesedeterminations find particular use in assessing the efficiency of mutantenzymes, as it is generally desired to produce variant enzymes thatexhibit greater ratios for particular substrates of interest. Forexample, the perhydrolase enzymes of the present invention are moreefficient in producing peracid from an ester substrate than enzymescurrently being used in cleaning, bleaching and disinfectingapplications. Another example of the present invention is a perhydrolasewith a lower activity on peracid degradation compared to the wild type.Another example of the present invention is a perhydrolase with higheractivity on more hydrophobic acyl groups than acetic acid. However, itis not intended that the present invention be limited to any particularsubstrate composition nor any specific substrate specificity.

As used herein, “surface property” is used in reference to anelectrostatic charge, as well as properties such as the hydrophobicityand/or hydrophilicity exhibited by the surface of a protein.

As used herein, the phrase “is independently selected from the groupconsisting of . . . ” means that moieties or elements that are selectedfrom the referenced Markush group can be the same, can be different orany mixture of elements as indicated in the following example:

A molecule having 3 R groups wherein each R group is independentlyselected from the group consisting of A, B and C. Here the three Rgroups may be: AAA, BBB, CCC, AAB, AAC, BBA, BBC, CCA, CCB, or ABC.

In reference to chemical compositions, the term “substituted” as usedherein, means that the organic composition or radical to which the termis applied is:

-   -   (a) made unsaturated by the elimination of at least one element        or radical; or    -   (b) at least one hydrogen in the compound or radical is replaced        with a moiety containing one or more (i) carbon, (ii)        oxygen, (iii) sulfur, (iv) nitrogen or (v) halogen atoms; or    -   (c) both (a) and (b).        Moieties which may replace hydrogen as described in (b)        immediately above, that contain only carbon and hydrogen atoms,        are hydrocarbon moieties including, but not limited to, alkyl,        alkenyl, alkynyl, alkyldienyl, cycloalkyl, phenyl, alkyl phenyl,        naphthyl, anthryl, phenanthryl, fluoryl, steroid groups, and        combinations of these groups with each other and with polyvalent        hydrocarbon groups such as alkylene, alkylidene and alkylidyne        groups. Moieties containing oxygen atoms that may replace        hydrogen as described in (b) immediately above include, but are        not limited to, hydroxy, acyl or keto, ether, epoxy, carboxy,        and ester containing groups. Moieties containing sulfur atoms        that may replace hydrogen as described in (b) immediately above        include, but are not limited to, the sulfur-containing acids and        acid ester groups, thioether groups, mercapto groups and        thioketo groups. Moieties containing nitrogen atoms that may        replace hydrogen as described in (b) immediately above include,        but are not limited to, amino groups, the nitro group, azo        groups, ammonium groups, amide groups, azido groups, isocyanate        groups, cyano groups and nitrile groups. Moieties containing        halogen atoms that may replace hydrogen as described in (b)        immediately above include chloro, bromo, fluoro, iodo groups and        any of the moieties previously described where a hydrogen or a        pendant alkyl group is substituted by a halo group to form a        stable substituted moiety.

It is understood that any of the above moieties (b)(i) through (b)(v)can be substituted into each other in either a monovalent substitutionor by loss of hydrogen in a polyvalent substitution to form anothermonovalent moiety that can replace hydrogen in the organic compound orradical.

As used herein, the terms “purified” and “isolated” refer to the removalof contaminants from a sample. For example, perhydrolases are purifiedby removal of contaminating proteins and other compounds within asolution or preparation that are not perhydrolases. In some embodiments,recombinant perhydrolases are expressed in bacterial or fungal hostcells and these recombinant perhydrolases are purified by the removal ofother host cell constituents; the percent of recombinant perhydrolasepolypeptides is thereby increased in the sample.

As used herein, “protein of interest,” refers to a protein (e.g., anenzyme or “enzyme of interest”) which is being analyzed, identifiedand/or modified. Naturally-occurring, as well as recombinant proteinsfind use in the present invention.

As used herein, “protein” refers to any composition comprised of aminoacids and recognized as a protein by those of skill in the art. Theterms “protein,” “peptide” and polypeptide are used interchangeablyherein. Wherein a peptide is a portion of a protein, those skilled inthe art understand the use of the term in context.

As used herein, functionally and/or structurally similar proteins areconsidered to be “related proteins.” In some embodiments, these proteinsare derived from a different genus and/or species, including differencesbetween classes of organisms (e.g., a bacterial protein and a fungalprotein). In some embodiments, these proteins are derived from adifferent genus and/or species, including differences between classes oforganisms (e.g., a bacterial enzyme and a fungal enzyme). In additionalembodiments, related proteins are provided from the same species.Indeed, it is not intended that the present invention be limited torelated proteins from any particular source(s). In addition, the term“related proteins” encompasses tertiary structural homologs and primarysequence homologs (e.g., the perhydrolase of the present invention). Infurther embodiments, the term encompasses proteins that areimmunologically cross-reactive. In most particularly preferredembodiments, the related proteins of the present invention very highratios of perhydrolysis to hydrolysis.

As used herein, the term “derivative” refers to a protein which isderived from a protein by addition of one or more amino acids to eitheror both the C- and N-terminal end(s), substitution of one or more aminoacids at one or a number of different sites in the amino acid sequence,and/or deletion of one or more amino acids at either or both ends of theprotein or at one or more sites in the amino acid sequence, and/orinsertion of one or more amino acids at one or more sites in the aminoacid sequence. The preparation of a protein derivative is preferablyachieved by modifying a DNA sequence which encodes for the nativeprotein, transformation of that DNA sequence into a suitable host, andexpression of the modified DNA sequence to form the derivative protein.

Related (and derivative) proteins comprise “variant proteins.” In somepreferred embodiments, variant proteins differ from a parent protein andone another by a small number of amino acid residues. The number ofdiffering amino acid residues may be one or more, preferably 1, 2, 3, 4,5, 10, 15, 20, 30, 40, 50, or more amino acid residues. In somepreferred embodiments, the number of different amino acids betweenvariants is between 1 and 10. In some particularly preferredembodiments, related proteins and particularly variant proteins compriseat least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 98%, or 99% amino acid sequence identity. Additionally, arelated protein or a variant protein as used herein, refers to a proteinthat differs from another related protein or a parent protein in thenumber of prominent regions. For example, in some embodiments, variantproteins have 1, 2, 3, 4, 5, or 10 corresponding prominent regions thatdiffer from the parent protein.

Several methods are known in the art that are suitable for generatingvariants of the perhydrolase enzymes of the present invention, includingbut not limited to site-saturation mutagenesis, scanning mutagenesis,insertional mutagenesis, random mutagenesis, site-directed mutagenesis,and directed-evolution, as well as various other recombinatorialapproaches.

In particularly preferred embodiments, homologous proteins areengineered to produce enzymes with the desired activity(ies). In someparticularly preferred embodiments, the engineered proteins are includedwithin the SGNH-hydrolase family of proteins. In some most preferredembodiments, the engineered proteins comprise at least one or acombination of the following conserved residues: L6, W14, W34, L38, R56,D62, L74, L78, H81, P83, M90, K97, G110, L114, L135, F180, G205. Inalternative embodiments, these engineered proteins comprise theGDSL-GRTT and/or ARTT motifs. In further embodiments, the enzymes aremultimers, including but not limited to dimers, octamers, and tetramers.In yet additional preferred embodiments, the engineered proteins exhibita perhydrolysis to hydrolysis ratio that is greater than 1.

An amino acid residue of a perhydrolase is equivalent to a residue of M.smegmatis perhydrolase if it is either homologous (i.e., having acorresponding position in either the primary and/or tertiary structure)or analogous to a specific residue or portion of that residue in M.smegmatis perhydrolase (i.e., having the same or similar functionalcapacity to combine, react, and/or chemically interact).

In some embodiments, in order to establish homology to primarystructure, the amino acid sequence of a perhydrolase is directlycompared to the M. smegmatis perhydrolase primary sequence andparticularly to a set of residues known to be invariant in allperhydrolases for which sequence is known. After aligning the conservedresidues, allowing for necessary insertions and deletions in order tomaintain alignment (i.e., avoiding the elimination of conserved residuesthrough arbitrary deletion and insertion), the residues equivalent toparticular amino acids in the primary sequence of M. smegmatisperhydrolase are defined. In preferred embodiments, alignment ofconserved residues conserves 100% of such residues. However, alignmentof greater than 75% or as little as 50% of conserved residues are alsoadequate to define equivalent residues. In preferred embodiments,conservation of the catalytic serine and histidine residues aremaintained. Conserved residues are used to define the correspondingequivalent amino acid residues of M. smegmatis perhydrolase in otherperhydrolases (e.g., perhydrolases from other Mycobacterium species, aswell as any other organisms).

In some embodiments of the present invention, the DNA sequence encodingM. smegmatis perhydrolase is modified. In some embodiments, thefollowing residues are modified: Cys7, Asp10, Ser11, Leu12, Thr13,Trp14, Trp16, Pro24, Thr25, Leu53, Ser54, Ala55, Thr64, Asp65, Arg67,Cys77, Thr91, Asn94, Asp95, Tyr99, Val125, Pro138, Leu140, Pro146,Pro148, Trp149, Phe150, Ile153, Phe154, Thr159, Thr186, Ile192, Ile194,and Phe196. However, it is not intended that the present invention belimited to sequence that are modified at these positions. Indeed, it isintended that the present invention encompass various modifications andcombinations of modifications.

In additional embodiments, equivalent residues are defined bydetermining homology at the level of tertiary structure for aperhydrolase whose tertiary structure has been determined by x-raycrystallography. In this context, “equivalent residues” are defined asthose for which the atomic coordinates of two or more of the main chainatoms of a particular amino acid residue of the carbonyl hydrolase andM. smegmatis perhydrolase (N on N, CA on CA, C on C, and O on O) arewithin 0.13 nm and preferably 0.1 nm after alignment. Alignment isachieved after the best model has been oriented and positioned to givethe maximum overlap of atomic coordinates of non-hydrogen protein atomsof the perhydrolase in question to the M. smegmatis perhydrolase. Asknown in the art, the best model is the crystallographic model givingthe lowest R factor for experimental diffraction data at the highestresolution available. Equivalent residues which are functionally and/orstructurally analogous to a specific residue of M. smegmatisperhydrolase are defined as those amino acids of the perhydrolases thatpreferentially adopt a conformation such that they either alter, modifyor modulate the protein structure, to effect changes in substratebinding and/or catalysis in a manner defined and attributed to aspecific residue of the M. smegmatis perhydrolase. Further, they arethose residues of the perhydrolase (in cases where a tertiary structurehas been obtained by x-ray crystallography), which occupy an analogousposition to the extent that although the main chain atoms of the givenresidue may not satisfy the criteria of equivalence on the basis ofoccupying a homologous position, the atomic coordinates of at least twoof the side chain atoms of the residue lie with 0.13 nm of thecorresponding side chain atoms of M. smegmatis perhydrolase. Thecoordinates of the three dimensional structure of M. smegmatisperhydrolase were determined and are set forth herein (See e.g., Example14) and find use as outlined above to determine equivalent residues onthe level of tertiary structure.

In some embodiments, some of the residues identified for substitution,insertion or deletion are conserved residues whereas others are not. Theperhydrolase mutants of the present invention include various mutants,including those encoded by nucleic acid that comprises a signalsequence. In some embodiments of perhydrolase mutants that are encodedby such a sequence are secreted by an expression host. In some furtherembodiments, the nucleic acid sequence comprises a homolog having asecretion signal.

Characterization of wild-type and mutant proteins is accomplished viaany means suitable and is preferably based on the assessment ofproperties of interest. For example, pH and/or temperature, as well asdetergent and/or oxidative stability is/are determined in someembodiments of the present invention. Indeed, it is contemplated thatenzymes having various degrees of stability in one or more of thesecharacteristics (pH, temperature, proteolytic stability, detergentstability, and/or oxidative stability) will find use. In still otherembodiments, perhydrolases with low peracid degradation activity areselected.

As used herein, “expression vector” refers to a DNA construct containinga DNA sequence that is operably linked to a suitable control sequencecapable of effecting the expression of the DNA in a suitable host. Suchcontrol sequences include a promoter to effect transcription, anoptional operator sequence to control such transcription, a sequenceencoding suitable mRNA ribosome binding sites and sequences whichcontrol termination of transcription and translation. The vector may bea plasmid, a phage particle, or simply a potential genomic insert. Oncetransformed into a suitable host, the vector may replicate and functionindependently of the host genome, or may, in some instances, integrateinto the genome itself. In the present specification, “plasmid,”“expression plasmid,” and “vector” are often used interchangeably as theplasmid is the most commonly used form of vector at present. However,the invention is intended to include such other forms of expressionvectors that serve equivalent functions and which are, or become, knownin the art.

In some preferred embodiments, the perhydrolase gene is ligated into anappropriate expression plasmid. The cloned perhydrolase gene is thenused to transform or transfect a host cell in order to express theperhydrolase gene. This plasmid may replicate in hosts in the sense thatit contains the well-known elements necessary for plasmid replication orthe plasmid may be designed to integrate into the host chromosome. Thenecessary elements are provided for efficient gene expression (e.g., apromoter operably linked to the gene of interest). In some embodiments,these necessary elements are supplied as the gene's own homologouspromoter if it is recognized, (i.e., transcribed, by the host), atranscription terminator (a polyadenylation region for eukaryotic hostcells) which is exogenous or is supplied by the endogenous terminatorregion of the perhydrolase gene. In some embodiments, a selection genesuch as an antibiotic resistance gene that enables continuous culturalmaintenance of plasmid-infected host cells by growth inantimicrobial-containing media is also included.

The following cassette mutagenesis method may be used to facilitate theconstruction of the perhydrolase variants of the present invention,although other methods may be used.

First, as described herein, a naturally-occurring gene encoding theperhydrolase is obtained and sequenced in whole or in part. Then, thesequence is scanned for a point at which it is desired to make amutation (deletion, insertion or substitution) of one or more aminoacids in the encoded perhydrolase. The sequences flanking this point areevaluated for the presence of restriction sites for replacing a shortsegment of the gene with an oligonucleotide pool which when expressedwill encode various mutants. Such restriction sites are preferablyunique sites within the protein gene so as to facilitate the replacementof the gene segment. However, any convenient restriction site which isnot overly redundant in the perhydrolase gene may be used, provided thegene fragments generated by restriction digestion can be reassembled inproper sequence. If restriction sites are not present at locationswithin a convenient distance from the selected point (from 10 to 15nucleotides), such sites are generated by substituting nucleotides inthe gene in such a fashion that neither the reading frame nor the aminoacids encoded are changed in the final construction. Mutation of thegene in order to change its sequence to conform to the desired sequenceis accomplished by M13 primer extension in accord with generally knownmethods. The task of locating suitable flanking regions and evaluatingthe needed changes to arrive at two convenient restriction sitesequences is made routine by the redundancy of the genetic code, arestriction enzyme map of the gene and the large number of differentrestriction enzymes. Note that if a convenient flanking restriction siteis available, the above method need be used only in connection with theflanking region which does not contain a site.

Once the naturally-occurring DNA and/or synthetic DNA is cloned, therestriction sites flanking the positions to be mutated are digested withthe cognate restriction enzymes and a plurality of endtermini-complementary oligonucleotide cassettes are ligated into thegene. The mutagenesis is simplified by this method because all of theoligonucleotides can be synthesized so as to have the same restrictionsites, and no synthetic linkers are necessary to create the restrictionsites.

As used herein, “corresponding to,” refers to a residue at theenumerated position in a protein or peptide, or a residue that isanalogous, homologous, or equivalent to an enumerated residue in aprotein or peptide.

As used herein, “corresponding region,” generally refers to an analogousposition along related proteins or a parent protein.

The terms “nucleic acid molecule encoding,” “nucleic acid sequenceencoding,” “DNA sequence encoding,” and “DNA encoding” refer to theorder or sequence of deoxyribonucleotides along a strand ofdeoxyribonucleic acid. The order of these deoxyribonucleotidesdetermines the order of amino acids along the polypeptide (protein)chain. The DNA sequence thus codes for the amino acid sequence.

As used herein, the term “analogous sequence” refers to a sequencewithin a protein that provides similar function, tertiary structure,and/or conserved residues as the protein of interest (i.e., typicallythe original protein of interest). For example, in epitope regions thatcontain an alpha helix or a beta sheet structure, the replacement aminoacids in the analogous sequence preferably maintain the same specificstructure. The term also refers to nucleotide sequences, as well asamino acid sequences. In some embodiments, analogous sequences aredeveloped such that the replacement amino acids result in a variantenzyme showing a similar or improved function. In some preferredembodiments, the tertiary structure and/or conserved residues of theamino acids in the protein of interest are located at or near thesegment or fragment of interest. Thus, where the segment or fragment ofinterest contains, for example, an alpha-helix or a beta-sheetstructure, the replacement amino acids preferably maintain that specificstructure.

As used herein, “homologous protein” refers to a protein (e.g.,perhydrolase) that has similar action and/or structure, as a protein ofinterest (e.g., an perhydrolase from another source). It is not intendedthat homologs be necessarily related evolutionarily. Thus, it isintended that the term encompass the same or similar enzyme(s) (i.e., interms of structure and function) obtained from different species. Insome preferred embodiments, it is desirable to identify a homolog thathas a quaternary, tertiary and/or primary structure similar to theprotein of interest, as replacement for the segment or fragment in theprotein of interest with an analogous segment from the homolog willreduce the disruptiveness of the change. In some embodiments, homologousproteins have induce similar immunological response(s) as a protein ofinterest.

As used herein, “homologous genes” refers to at least a pair of genesfrom different species, which genes correspond to each other and whichare identical or very similar to each other. The term encompasses genesthat are separated by speciation (i.e., the development of new species)(e.g., orthologous genes), as well as genes that have been separated bygenetic duplication (e.g., paralogous genes). These genes encode“homologous proteins.”

As used herein, “ortholog” and “orthologous genes” refer to genes indifferent species that have evolved from a common ancestral gene (i.e.,a homologous gene) by speciation. Typically, orthologs retain the samefunction during the course of evolution. Identification of orthologsfinds use in the reliable prediction of gene function in newly sequencedgenomes.

As used herein, “paralog” and “paralogous genes” refer to genes that arerelated by duplication within a genome. While orthologs retain the samefunction through the course of evolution, paralogs evolve new functions,even though some functions are often related to the original one.Examples of paralogous genes include, but are not limited to genesencoding trypsin, chymotrypsin, elastase, and thrombin, which are allserine proteinases and occur together within the same species.

As used herein, “wild-type” and “native” proteins are those found innature. The terms “wild-type sequence,” and “wild-type gene” are usedinterchangeably herein, to refer to a sequence that is native ornaturally occurring in a host cell. In some embodiments, the wild-typesequence refers to a sequence of interest that is the starting point ofa protein engineering project. The genes encoding thenaturally-occurring protein may be obtained in accord with the generalmethods known to those skilled in the art. The methods generallycomprise synthesizing labeled probes having putative sequences encodingregions of the protein of interest, preparing genomic libraries fromorganisms expressing the protein, and screening the libraries for thegene of interest by hybridization to the probes. Positively hybridizingclones are then mapped and sequenced.

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule that is comprised of segments of DNA joined together by meansof molecular biological techniques.

The term “recombinant oligonucleotide” refers to an oligonucleotidecreated using molecular biological manipulations, including but notlimited to, the ligation of two or more oligonucleotide sequencesgenerated by restriction enzyme digestion of a polynucleotide sequence,the synthesis of oligonucleotides (e.g., the synthesis of primers oroligonucleotides) and the like.

The degree of homology between sequences may be determined using anysuitable method known in the art (See e.g., Smith and Waterman, Adv.Appl. Math., 2:482 [1981]; Needleman and Wunsch, J. Mol. Biol., 48:443[1970]; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988];programs such as GAP, BESTFIT, FAS TA, and TFASTA in the WisconsinGenetics Software Package (Genetics Computer Group, Madison, Wis.); andDevereux et al., Nucl. Acid Res., 12:387-395 [1984]).

For example, PILEUP is a useful program to determine sequence homologylevels. PILEUP creates a multiple sequence alignment from a group ofrelated sequences using progressive, pairwise alignments. It can alsoplot a tree showing the clustering relationships used to create thealignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng and Doolittle, (Feng and Doolittle, J. Mol. Evol.,35:351-360 [1987]). The method is similar to that described by Higginsand Sharp (Higgins and Sharp, CABIOS 5:151-153 [1989]). Useful PILEUPparameters including a default gap weight of 3.00, a default gap lengthweight of 0.10, and weighted end gaps. Another example of a usefulalgorithm is the BLAST algorithm, described by Altschul et al.,(Altschul et al., J. Mol. Biol., 215:403-410, [1990]; and Karlin et al.,Proc. Natl. Acad. Sci. USA 90:5873-5787 [1993]). One particularly usefulBLAST program is the WU-BLAST-2 program (See, Altschul et al., Meth.Enzymol., 266:460-480 [1996]). parameters “W,” “T,” and “X” determinethe sensitivity and speed of the alignment. The BLAST program uses asdefaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (See,Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 [1989])alignments (B) of 50, expectation (E) of 10, M′5, N′-4, and a comparisonof both strands.

As used herein, “percent (%) nucleic acid sequence identity” is definedas the percentage of nucleotide residues in a candidate sequence thatare identical with the nucleotide residues of the sequence.

As used herein, the term “hybridization” refers to the process by whicha strand of nucleic acid joins with a complementary strand through basepairing, as known in the art.

As used herein, the phrase “hybridization conditions” refers to theconditions under which hybridization reactions are conducted. Theseconditions are typically classified by degree of “stringency” of theconditions under which hybridization is measured. The degree ofstringency can be based, for example, on the melting temperature (Tm) ofthe nucleic acid binding complex or probe. For example, “maximumstringency” typically occurs at about Tm-5° C. (5° below the Tm of theprobe); “high stringency” at about 5-10° below the Tm; “intermediatestringency” at about 10-20° below the Tm of the probe; and “lowstringency” at about 20-25° below the Tm. Alternatively, or in addition,hybridization conditions can be based upon the salt or ionic strengthconditions of hybridization and/or one or more stringency washes. Forexample, 6×SSC=very low stringency; 3×SSC=low to medium stringency;1×SSC=medium stringency; and 0.5×SSC=high stringency. Functionally,maximum stringency conditions may be used to identify nucleic acidsequences having strict identity or near-strict identity with thehybridization probe; while high stringency conditions are used toidentify nucleic acid sequences having about 80% or more sequenceidentity with the probe.

For applications requiring high selectivity, it is typically desirableto use relatively stringent conditions to form the hybrids (e.g.,relatively low salt and/or high temperature conditions are used).

The phrases “substantially similar and “substantially identical” in thecontext of at least two nucleic acids or polypeptides typically meansthat a polynucleotide or polypeptide comprises a sequence that has atleast about 40% identity, more preferable at least about 50% identity,yet more preferably at least about 60% identity, preferably at leastabout 75% identity, more preferably at least about 80% identity, yetmore preferably at least about 90%, still more preferably about 95%,most preferably about 97% identity, sometimes as much as about 98% andabout 99% sequence identity, compared to the reference (i.e., wild-type)sequence. Sequence identity may be determined using known programs suchas BLAST, ALIGN, and CLUSTAL using standard parameters. (See e.g.,Altschul, et al., J. Mol. Biol. 215:403-410 [1990]; Henikoff et al.,Proc. Natl. Acad. Sci. USA 89:10915 [1989]; Karin et al., Proc. Natl.Acad. Sci. USA 90:5873 [1993]; and Higgins et al., Gene 73:237-244[1988]). Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information. Also,databases may be searched using FASTA (Pearson et al., Proc. Natl. Acad.Sci. USA 85:2444-2448 [1988]). One indication that two polypeptides aresubstantially identical is that the first polypeptide is immunologicallycross-reactive with the second polypeptide. Typically, polypeptides thatdiffer by conservative amino acid substitutions are immunologicallycross-reactive. Thus, a polypeptide is substantially identical to asecond polypeptide, for example, where the two peptides differ only by aconservative substitution. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions (e.g., within a rangeof medium to high stringency).

As used herein, “equivalent residues” refers to proteins that shareparticular amino acid residues. For example, equivalent resides may beidentified by determining homology at the level of tertiary structurefor a protein (e.g., perhydrolase) whose tertiary structure has beendetermined by x-ray crystallography. Equivalent residues are defined asthose for which the atomic coordinates of two or more of the main chainatoms of a particular amino acid residue of the protein having putativeequivalent residues and the protein of interest (N on N, CA on CA, C onC and O on O) are within 0.13 nm and preferably 0.1 nm after alignment.Alignment is achieved after the best model has been oriented andpositioned to give the maximum overlap of atomic coordinates ofnon-hydrogen protein atoms of the proteins analyzed. The preferred modelis the crystallographic model giving the lowest R factor forexperimental diffraction data at the highest resolution available,determined using methods known to those skilled in the art ofcrystallography and protein characterizationanalysis.

As used herein, the terms “hybrid perhydrolases” and “fusionperhydrolases” refer to proteins that are engineered from at least twodifferent or “parental” proteins. In preferred embodiments, theseparental proteins are homologs of one another. For example, in someembodiments, a preferred hybrid perhydrolase or fusion protein containsthe N-terminus of a protein and the C-terminus of a homolog of theprotein. In some preferred embodiment, the two terminal ends arecombined to correspond to the full-length active protein.

The term “regulatory element” as used herein refers to a genetic elementthat controls some aspect of the expression of nucleic acid sequences.For example, a promoter is a regulatory element which facilitates theinitiation of transcription of an operably linked coding region.Additional regulatory elements include splicing signals, polyadenylationsignals and termination signals.

As used herein, “host cells” are generally prokaryotic or eukaryotichosts which are transformed or transfected with vectors constructedusing recombinant DNA techniques known in the art. Transformed hostcells are capable of either replicating vectors encoding the proteinvariants or expressing the desired protein variant. In the case ofvectors which encode the pre- or prepro-form of the protein variant,such variants, when expressed, are typically secreted from the host cellinto the host cell medium.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means transformation, transduction ortransfection. Means of transformation include protoplast transformation,calcium chloride precipitation, electroporation, naked DNA and the likeas known in the art. (See, Chang and Cohen, Mol. Gen. Genet.,168:111-115 [1979]; Smith et al., Appl. Env. Microbiol., 51:634 [1986];and the review article by Ferrari et al., in Harwood, Bacillus, PlenumPublishing Corporation, pp. 57-72 [1989]).

The term “promoter/enhancer” denotes a segment of DNA which containssequences capable of providing both promoter and enhancer functions (forexample, the long terminal repeats of retroviruses contain both promoterand enhancer functions). The enhancer/promoter may be “endogenous” or“exogenous” or “heterologous.” An endogenous enhancer/promoter is onewhich is naturally linked with a given gene in the genome. An exogenous(heterologous) enhancer/promoter is one which is placed in juxtapositionto a gene by means of genetic manipulation (i.e., molecular biologicaltechniques).

The presence of “splicing signals” on an expression vector often resultsin higher levels of expression of the recombinant transcript. Splicingsignals mediate the removal of introns from the primary RNA transcriptand consist of a splice donor and acceptor site (Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, New York [1989], pp. 16.7-16.8). A commonly usedsplice donor and acceptor site is the splice junction from the 16S RNAof SV40.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell whichhas stably integrated foreign or exogenous DNA into the genomic DNA ofthe transfected cell.

The terms “selectable marker” or “selectable gene product” as usedherein refer to the use of a gene which encodes an enzymatic activitythat confers resistance to an antibiotic or drug upon the cell in whichthe selectable marker is expressed.

As used herein, the terms “amplification” and “gene amplification” referto a process by which specific DNA sequences are disproportionatelyreplicated such that the amplified gene becomes present in a higher copynumber than was initially present in the genome. In some embodiments,selection of cells by growth in the presence of a drug (e.g., aninhibitor of an inhibitable enzyme) results in the amplification ofeither the endogenous gene encoding the gene product required for growthin the presence of the drug or by amplification of exogenous (i.e.,input) sequences encoding this gene product, or both. Selection of cellsby growth in the presence of a drug (e.g., an inhibitor of aninhibitable enzyme) may result in the amplification of either theendogenous gene encoding the gene product required for growth in thepresence of the drug or by amplification of exogenous (i.e., input)sequences encoding this gene product, or both.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

As used herein, the term “co-amplification” refers to the introductioninto a single cell of an amplifiable marker in conjunction with othergene sequences (i.e., comprising one or more non-selectable genes suchas those contained within an expression vector) and the application ofappropriate selective pressure such that the cell amplifies both theamplifiable marker and the other, non-selectable gene sequences. Theamplifiable marker may be physically linked to the other gene sequencesor alternatively two separate pieces of DNA, one containing theamplifiable marker and the other containing the non-selectable marker,may be introduced into the same cell.

As used herein, the terms “amplifiable marker,” “amplifiable gene,” and“amplification vector” refer to a marker, gene or a vector encoding agene which permits the amplification of that gene under appropriategrowth conditions.

As used herein, the term “amplifiable nucleic acid” refers to nucleicacids which may be amplified by any amplification method. It iscontemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample which is analyzed for the presence of “target”(defined below). In contrast, “background template” is used in referenceto nucleic acid other than sample template which may or may not bepresent in a sample. Background template is most often inadvertent. Itmay be the result of carryover, or it may be due to the presence ofnucleic acid contaminants sought to be purified away from the sample.For example, nucleic acids from organisms other than those to bedetected may be present as background in a test sample.

“Template specificity” is achieved in most amplification techniques bythe choice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogeneous mixture of nucleic acid. For example, inthe case of Qβ replicase, MDV-1 RNA is the specific template for thereplicase (See e.g., Kacian et al., Proc. Natl. Acad. Sci. USA 69:3038[1972]). Other nucleic acids are not replicated by this amplificationenzyme. Similarly, in the case of T7 RNA polymerase, this amplificationenzyme has a stringent specificity for its own promoters (See,Chamberlin et al., Nature 228:227 [1970]). In the case of T4 DNA ligase,the enzyme will not ligate the two oligonucleotides or polynucleotides,where there is a mismatch between the oligonucleotide or polynucleotidesubstrate and the template at the ligation junction (See, Wu andWallace, Genomics 4:560 [1989]). Finally, Taq and Pfu polymerases, byvirtue of their ability to function at high temperature, are found todisplay high specificity for the sequences bounded and thus defined bythe primers; the high temperature results in thermodynamic conditionsthat favor primer hybridization with the target sequences and nothybridization with non-target sequences.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, which is capable of hybridizing to anotheroligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the present invention will be labeled with any “reportermolecule,” so that is detectable in any detection system, including, butnot limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, and luminescentsystems. It is not intended that the present invention be limited to anyparticular detection system or label.

As used herein, the term “target,” when used in reference toamplification methods (e.g., the polymerase chain reaction), refers tothe region of nucleic acid bounded by the primers used for polymerasechain reaction. Thus, the “target” is sought to be sorted out from othernucleic acid sequences. A “segment” is defined as a region of nucleicacid within the target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe methods of U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188,hereby incorporated by reference, which include methods for increasingthe concentration of a segment of a target sequence in a mixture ofgenomic DNA without cloning or purification. This process for amplifyingthe target sequence consists of introducing a large excess of twooligonucleotide primers to the DNA mixture containing the desired targetsequence, followed by a precise sequence of thermal cycling in thepresence of a DNA polymerase. The two primers are complementary to theirrespective strands of the double stranded target sequence. To effectamplification, the mixture is denatured and the primers then annealed totheir complementary sequences within the target molecule. Followingannealing, the primers are extended with a polymerase so as to form anew pair of complementary strands. The steps of denaturation, primerannealing and polymerase extension can be repeated many times (i.e.,denaturation, annealing and extension constitute one “cycle”; there canbe numerous “cycles”) to obtain a high concentration of an amplifiedsegment of the desired target sequence. The length of the amplifiedsegment of the desired target sequence is determined by the relativepositions of the primers with respect to each other, and therefore, thislength is a controllable parameter. By virtue of the repeating aspect ofthe process, the method is referred to as the “polymerase chainreaction” (hereinafter “PCR”). Because the desired amplified segments ofthe target sequence become the predominant sequences (in terms ofconcentration) in the mixture, they are said to be “PCR amplified”.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCTPor dATP, into the amplified segment). In addition to genomic DNA, anyoligonucleotide or polynucleotide sequence can be amplified with theappropriate set of primer molecules. In particular, the amplifiedsegments created by the PCR process itself are, themselves, efficienttemplates for subsequent PCR amplifications.

As used herein, the terms “PCR product,” “PCR fragment,” and“amplification product” refer to the resultant mixture of compoundsafter two or more cycles of the PCR steps of denaturation, annealing andextension are complete. These terms encompass the case where there hasbeen amplification of one or more segments of one or more targetsequences.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

The Present Invention

In some most particularly preferred embodiments, the present inventionfinds use in the enzymatic generation of peracids from ester substratesand hydrogen peroxide. In some preferred embodiments, the substrates areselected from one or more of the following: formic acid, acetic acid,propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid,nonanoic acid, decanoic acid, dodecanoic acid, myristic acid, palmiticacid, stearic acid, and oleic acid. Importantly, the present inventionprovides means for effective cleaning, bleaching, and disinfecting overbroad pH and temperature ranges. In some embodiments, the pH rangeutilized in this generation is 4-12. In alternative embodiments, thetemperature range utilized is between 5° and 90° C. The presentinvention provides advantages over the presently used systems (See e.g.,EP Appln. 87-304933.9) in that bleaching is possible at the optimum pHof peracid oxidation, as well as providing bleaching at neutral pH,acidic pHs, and at low temperatures. While the present invention isdescribed herein most fully in regard to laundry and fabric care, it isnot intended that the present invention be limited to theseapplications. Indeed, the present invention finds use in varioussettings, particularly those in which bleaching by peracids and/orhydrogen peroxide are desired, including but not limited to laundry,fabric treatment, pulp and paper processing, personal care applications,disinfection and cleaning of hard surfaces. For example, it iscontemplated that the compositions of the present invention will finduse in bleaching of pulp, including use in methods such as those setforth in U.S. Pat. Nos. 6,569,286, 5,785,812, 6,165,318, and 4,400,237,all of which are herein incorporated by reference.

Historically, sodium perborate, and more recently, sodium percarbonate,have been used as bleaching compounds, particularly in European laundrydetergents. This compound decomposes rapidly in aqueous solution toyield hydrogen peroxide (H₂O₂), which is the active bleaching species.As sodium perborate is more active at temperatures above 80° C., andless active in the temperature range of 40-60° C. (i.e., washtemperatures that have become most commonly preferred as of the 1950s),bleaching activators have been incorporated into laundry detergents thatcontain sodium perborate. Indeed, most laundry detergents containbleaching activators. These activators are compounds with O- orN-bounded acetyl groups that are able to react with the stronglynucleophilic hydroperoxy anion to yield peroxyacetic acid. Since thereacting species is hydroperoxy anion, alkaline pHs are essential forthe efficient conversion of these activators to peracids. Theperoxyacetic acid is decomposed in weakly basic media to form singletoxygen (See, Hofmann et al., J. Prakt. Chem., 334:293-297 [1992]).

Hydrogen peroxide is a particularly effective bleach at hightemperatures (e.g., >40° C.) and pH (>10), conditions that are typicallyused in washing fabrics in some settings. However, as indicated above,cold water washing is becoming more commonly used and results in lesseffective bleaching by H₂O₂ than use of hot water. To overcome this lowtemperature disadvantage, detergent formulations typically includebleach boosters, such as TAED (N,N,N′N′-tetraacetylethylenediamine),NOBS (nonanoyloxybenzene sulfonate), etc. These boosters combine withH₂O₂ to form peracetic acid, a peracid species that is more effectivethan H₂O₂ alone. Although it helps the bleaching capability ofdetergent, the TAED reaction is only approximately 50% efficient, asonly two out of the four acetyl groups in TAED are converted toperacids. Additionally, conversion of TAED into peracetic acid byhydrogen peroxide is efficient only at alkaline pHs and hightemperatures. Thus, the TAED reaction is not optimized for use in allbleaching applications (e.g., those involving neutral or acidic pHs, andcold water). The present invention provides means to overcome thedisadvantages of TAED use. For example, the present invention finds usein cold water applications, as well as those involving neutral or acidicpH levels. Furthermore, the present invention provides means for peracidgeneration from hydrogen peroxide, with a high perhydrolysis tohydrolysis ratio. The present invention further provides advantages overcompositions that contain enzymes such as esterases and lipases) whichhave very low perhydrolysis to hydrolysis ratios.

In addition to its applications in detergents, the present inventionprovides methods and compositions for the use of peracids in textilebleaching and in various other applications. In some embodiments, thepresent invention provides one-step methods for textile processingapplications, including but not limited to one-step desizing, scouringand bleaching processes (See e.g., EP WO 03002810, EP 1255888, WO0164993, and US 20020007516, all of which are hereby incorporated byreference). As described in greater detail herein, in some embodiments,bleaching involves processing textile material before it is dyed and/orafter it is incorporated into textile goods. However, it is not intendedthat the present invention be limited to any particular regimen of usenor any particular textile material.

Furthermore, the peracetic technology of the present invention finds useas an effective bactericide (See, Baldry, J. Appl. Bacteriol.,54:417-423 [1983]). Thus, the present invention provides compositionsand methods for the sterilization/disinfection of various objects,including but not limited to medical devices, medical equipment,industrial equipment, and fermenters, as well as any additional objectthat needs to be sterilized or disinfected. As discussed in greaterdetail below, during the development of the present invention, theenzyme of the present invention was used in a standard cell killexperiment to demonstrate this suitability. In additional embodiments,the present invention provides compositions and methods suitable for usein biofilm control, such as in cooling towers.

Also as described in more detail in the Examples below, the presentinvention provides many advantages for cleaning and/or sterilization ofa wide range of objects, including but not limited to clothing, fabrics,medical devices, etc. In addition, the present invention providescompositions that are effective in cleaning, bleaching, anddisinfecting, over a range of wash temperatures and pHs. In additionalembodiments, the present invention finds use in degradation of peracidsthrough the perhydrolase peracid degradation activity. In some preferredembodiments, this activity is used in peracid waste clean upapplications.

Furthermore, the perhydrolase enzymes of the present invention areactive on various acyl donor substrates, as well as being active at lowsubstrate concentrations, and provide means for efficient perhydrolysisdue to the high peracid:acid ratio. Indeed, it has been recognized thathigher perhydrolysis to hydrolysis ratios are preferred for bleachingapplications (See e.g., U.S. Pat. Nos. 5,352,594, 5,108,457, 5,030,240,3,974,082, and 5,296,616, all of which are herein incorporated byreference). In preferred embodiments, the perhydrolase enzymes of thepresent invention provide perhydrolysis to hydrolysis ratios that aregreater than 1. In particularly preferred embodiments, the perhydrolaseenzymes provide a perhydrolysis to hydrolysis ratio greater than 1 andare find use in bleaching.

In addition, it has been shown to be active in commonly used detergentformulations (e.g., Aria Futur, WOB, etc.). Thus, the present inventionprovides many advantages in various cleaning settings.

As indicated above, key components to peracid production by enzymaticperhydrolysis are enzyme, ester substrate, and hydrogen peroxide.Hydrogen peroxide can be either added directly in batch, or generatedcontinuously “in situ.” Current washing powders use batch additions ofH₂O₂, in the form of percarbonate or perborate salts that spontaneouslydecompose to H₂O₂. The perhydrolase enzymes of the present inventionfind use in the same washing powder batch method as the H₂O₂ source.However, these enzymes also find use with any other suitable source ofH₂O₂, including that generated by chemical, electro-chemical, and/orenzymatic means. Examples of chemical sources are the percarbonates andperborates mentioned above, while an example of an electrochemicalsource is a fuel cell fed oxygen and hydrogen gas, and an enzymaticexample includes production of H₂O₂ from the reaction of glucose withglucose oxidase. The following equation provides an example of a coupledsystem that finds use with the present invention.

It is not intended that the present invention be limited to any specificenzyme, as any enzyme that generates H₂O₂ with a suitable substratefinds use in the methods of the present invention. For example, lactateoxidases from Lactobacillus species which are known to create H₂O₂ fromlactic acid and oxygen find use with the present invention. Indeed, oneadvantage of the methods of the present invention is that the generationof acid (e.g., gluconic acid in the above example) reduces the pH of abasic solution to the pH range in which the peracid is most effective inbleaching (i.e., at or below the pKa). Other enzymes (e.g., alcoholoxidase, ethylene glycol oxidase, glycerol oxidase, amino acid oxidase,etc.) that can generate hydrogen peroxide also find use with estersubstrates in combination with the perhydrolase enzymes of the presentinvention to generate peracids. In some preferred embodiments, the estersubstrates are selected from one or more of the following acids: formicacid, acetic acid, propionic acid, butyric acid, valeric acid, caproicacid, caprylic acid, nonanoic acid, decanoic acid, dodecanoic acid,myristic acid, palmitic acid, stearic acid, and oleic acid. Thus, asdescribed herein, the present invention provides definite advantagesover the currently used methods and compositions for detergentformulation and use, as well as various other applications.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention provides methods and compositions comprising atleast one perhydrolase enzyme for cleaning and other applications. Insome particularly preferred embodiments, the present invention providesmethods and compositions for generation of peracids. The presentinvention finds particular use in applications involving cleaning,bleaching and disinfecting.

Cloning and Characterization of M. smegmatis Perhydrolase

The cloning of the M. smegmatis perhydrolase (i.e., referred to hereinas the “phd” gene, which encodes the “Phd” protein; this perhydrolasegene is sometimes herein referred to as the “act” gene and the proteinis sometimes referred to as the “Act” protein) of the present inventionwas based on peptide sequence data from the acyltransferase purifiedfrom Mycobacterium parafortuitum (previously known as Corynebacteriumoxydans) and published information regarding the 7-aminocephalosporanicacid (7-ACA) arylesterase gene of Agrobacterium radiobacter (Sakai etal., J. Ferment. Bioengineer., 85: 138-143 [1998]). Two peptidesequences from purified M. parafortuitum acyltransferase were found tobe similar to internal N- and C-terminal regions of the A. radiobacter7-ACA-arylesterase (47% and 42% identity respectively).

A set of PCR primers was designed based on the amino acid sequence ofthese internal peptides (designated “AtintF” and “AtintR”). Another setof primers was developed based on the 5′ and 3′ ends (“ATNcoI” and“ATBamH1”) of the A. radiobacter 7-ACA DNA sequence. A single product ofthe expected size was amplified from M. parafortuitum chromosomal DNAusing both sets of primers. The full length product, amplified by theATNcoI/ATBamH1 primer pair, was cloned into pET16b and transformed intoBL21 cells (Novagen, Madison, Wis.). This clone had a sequence identicalto that of the A. radiobacter 7-ACA gene. As it was determined thatpurified M. parafortuitum perhydrolase was not the 7-ACA acyl esterase,it was concluded that this was not the gene encoding the perhydrolase ofthe present invention.

Thus, efforts were further focused on M. smegmatis for cloning andexpression of the perhydrolase of the present invention. To identify theM. parafortuitum gene based on enzyme activity screening, a plasmidlibrary of M. parafortuitum DNA in M. smegmatis was constructed using aplasmid with a promoter to drive expression of cloned genes.Surprisingly, M. smegmatis itself was found to be positive forperhydrolase and acyltransferase activity. Thus, in some instancesherein, the perhydrolase is referred to as “ACT” (or “Act”). A proteinBLAST search of the M. smegmatis unfinished genome using the sequence ofthe A. radiobacter 7-ACA identified a 2 kb contig containing an ORF(open reading frame) that encoded a hypothetical protein that wassimilar but not identical to the 7-ACA protein. Based on this sequence,primers were designed and used to amplify the gene from M. smegmatis(ATCC 10143). By adding an E. coli ribosome binding site upstream of thestart codon, a clone that expressed active enzyme was obtained. Thevector used was either pCR2.1TOPO or pBluntIITOPO (Invitrogen, Carlsbad,Calif.), in E. coli Top10 cells. The gene was expressed constitutivelyfrom the plasmid-encoded lac promoter. This enzyme carried out the samereactions as the originally described M. parafortuitum acyltransferase.

During the characterization of the perhydrolase of the presentinvention, standard protein BLAST searches identified a few proteins(<20) with sequence similarity of 30-80%. This group included the 7-ACAarylesterases from A. radiobacter and other organisms, which have 43%identity with M. smegmatis perhydrolase. All of the identified homologswith at least 40% similarity have a GDS motif very near the N-terminalend. All of the proteins also contain most of the conserved residueswhich could place them within the suggested GDSL family of lipolyticenzymes (See e.g., Upton and Buckley, Trends Biochem. Sci., 20:178[1995]). However, enzymes mentioned in this paper do not appear onhomology searches with the perhydrolase protein. Indeed these proteinshave less than 20% similarity with the perhydrolase and its homologs,suggesting that the acyltransferase-related (and perhydrolase of thepresent invention) enzymes form a subfamily.

The natural function of the enzyme of the present invention and theclosely related proteins, apart from the 7-ACA arylesterase, have notbeen biochemically determined M. smegmatis appears to be the onlyorganism with the acyltransferase/perhydrolase in an operon with aputative penicillin binding protein (PBP). While it is not intended thatthe present invention be limited to any particular mechanism, thissuggests that the enzyme may be involved in cell wallsynthesis/structure or modification of molecules taken up from theenvironment. There are no homologues of the perhydrolase of the presentinvention that have been identified in M. tuberculosis or M. leprae todate. However, some organisms were determined to have multiplehomologues (e.g., S. meliloti).

During the development of the present invention, various mutations weremade in the M. smegmatis perhydrolase in order to assess its activity.This enzyme contains two cysteine residues, which were hypothesized aspotentially forming disulfide bonds, both of which were changed toalanine, in order to determine whether or not the C residues had anyeffect on the activity of the enzyme. Activity assay results obtainedusing the transesterification (in aqueous solution) assay describedherein indicated that C7A, as well as C77A, and a double mutant (C7A andC77A) were of the same size and specific activity.

Many enzymes have the amino acid serine as part of their active site andare therefore referred to, among other designations, as “serinehydrolases.” The active site may consist of a catalytic triad of S(serine), D (aspartic acid) and H (histidine). Examples of such enzymesinclude, but are not limited to subtilisin (D32-H64-S215), chymotrypsin(H57-D102-5195) and lipases in the alpha/beta hydrolase family (e.g.,S126-D176-H206). A typical motif for lipases is the GDSL motif (Uptonand Buckley, supra [1995]) in which the S is the active site serine.Since the perhydrolase of the present invention was determined to have aGDSL (amino acids 9-12) motif, the S11 was mutated to an A, in order toconfirm the involvement of this S in the active site. As indicated inthe Examples, the activity assay results indicated that S11A had only 1%of the activity of the wild-type enzyme. Deletion of the C-terminal 25amino acids also resulted in abrogation of the activity, suggesting thatthese amino acids either contained a residue involved directly in theactive site, and/or that the structure of the protein was affected suchthat the active site was no longer able to catalyze the reactions. Inaddition, the predicted active site residues, D192 and H195 were mutatedto A. Neither mutant had activity, confirming that the active siteresidues of the perhydrolase of the present invention consist of S11,D192 and H195. However, it is not intended that the present invention belimited to any particular mechanism, nor is the present inventionlimited to mutation(s) at any particular active site residues.

Cloning of M. parafortuitum Perhydrolase

There were some differences between the N-terminal peptide sequenceobtained from the M. parafortuitum enzyme and the N-terminal sequence ofM. smegmatis perhydrolase. However, there was a sequence in theC-terminal region of the M. smegmatis perhydrolase identical to theC-terminal peptide sequence of the M. parafortuitum enzyme. Two primerswere designed to amplify a partial sequence of the M. parafortuitumperhydrolase gene; the sequence of the reverse primer was identical tothe sequence of the corresponding region in M. smegmatis perhydrolasegene, and the sequence of the forward primer was based on M. smegmatiscodon usage. The forward primer, MP5:5′-ATGGGTACCCGACGAATTCTGTCCTTCGGTGATTCCCTGACCT-3′ (SEQ ID NO:11) and thereverse primer MPC-intR5′-GATTCCGTCGACGCCGTCGGTGCTGATCACCGAACCCGCGTCGAAGAACGG-3′ (SEQ IDNO:12). The partial gene was amplified from the chromosome of M.parafortuitum and cloned into pCR2.1TOPO (Invitrogen, Carlsbad, Calif.).Sequence analysis showed that the enzyme is very similar, but notidentical to the M. smegmatis perhydrolase (77% identity). Based on themolecular weights of the monomers of the perhydrolases determined bySDS-PAGE (MP AT: 26 kDa, MSAT: 24 kDa, MP cloned AT: ˜18 kDa), the clonefrom primers made to the internal fragment was determined to be missingapproximately 70 amino acids (˜8 kDa). The remaining sequence at the5′-end of the M. parafortuitum gene can be obtained by any of severalmethods suitable and familiar to those skilled in the art of molecularbiology, including, but not limited to, inverse PCR, probing ofplasmid/cosmid libraries of M. parafortuitum chromosomal DNA, sequencingof the gene directly from chromosomal DNA (e.g., as performed byFidelity Systems, Bethesda Md.).

Expression of the M. smegmatis Perhydrolase

The perhydrolase is an intracellular protein in its native host.Production of the perhydrolase in non-native hosts may also be doneintracellularly. However, in some embodiments, a signal sequence isadded to the perhydrolase, which facilitates expression of theperhydrolase by secretion into the periplasm (i.e., in Gram-negativeorganisms, such as E. coli), or into the extracellular space (i.e., inGram-positive organisms, such as Bacillus and Actinomycetes), oreukaryotic hosts (e.g., Trichoderma, Aspergillus, Saccharomyces, andPichia). Of course, these are just a few examples of possibleprokaryotic and eukaryotic hosts. It is not intended that the presentinvention be limited to these specific hosts, as various other organismsfind use as expression hosts in the present invention.

A variety of commercially available expression systems, including butnot limited to pBAD, plac, T7, find use in the expression of theperhydrolase in Gram-negative hosts (e.g., E. coli). In someembodiments, the same types of promoters find use in anotherGram-negative host, Pantoea citrea.

To test expression in E. coli two strategies were used: 1) adding an RBS(ribosome binding site) to the 5′ end of the phd gene and cloning thegene into pCRBLUNTIITOPO (Invitrogen), thus allowing expression directlyfrom the pLac promoter available in that vector; and 2) cloning the phdgene under control of the T7 promoter in the plasmid pET16b (Novagen).In the latter system, expression of the gene is inducible by addition ofIPTG to the growing culture and use of a specific host cell (e.g.,BL21(λDE3)pLysS (Novagen)) that contains the 2DE3 lysogen encoding theT7 RNA polymerase. The first strategy produces a plasmid capable ofallowing expression of the perhydrolase protein in other Gram-negativehosts (e.g., P. citrea).

To express protein in E. coli or P. citrea using the first strategy,cultures were grown from single, purified colonies at 37° C. overnightin L broth plus the appropriate antibiotic (example, kanamycin 50μg/ml). Expression of the protein was determined by the pNB assay (See,Example 1) after lysis of the cells.

Expression of the perhydrolase using the T7 expression system requiresinduction of the culture with the addition of IPTG (e.g., 100 mmole IPTGadded at an OD₅₅₀ of 0.4). Overnight cultures, inoculated from a singlecolony, are used to inoculate the expression culture of the desiredvolume (25 mls to several liters) at an OD₅₅₀ of 0.1. The expressionculture was then grown at the desired temperature (e.g., 25° C., 30° C.,37° C.) until an OD₅₅₀ of 0.4 was reached, after which IPTG was added.Expression was allowed to continue for 3 hours to overnight. Proteinexpression was monitored by pNB activity assay as described inExample 1. Usually, expression from the T7 system gives a high titer ofprotein, sufficient for further analysis such as crystallography.

Bacillus species are well-known as suitable hosts for expression ofextracellular proteins (e.g., proteases). Intracellular expression ofproteins is less well known. Expression of the perhydrolase proteinintracellularly in Bacillus subtilis can be done using a variety ofpromoters, including, but not limited to pVeg, pSPAC, pAprE, or pAmyE inthe absence of a signal sequence on the 5′ end of the gene. In someembodiments, expression is achieved from a replicating plasmid (high orlow copy number), while in alternative embodiments, expression isachieved by integrating the desired construct into the chromosome.Integration can be done at any locus, including but not limited to theaprE, amyE, or pps locus. In some embodiments, the perhydrolase isexpressed from one or more copies of the integrated construct. Inalternative embodiments, multiple integrated copies are obtained by theintegration of a construct capable of amplification (e.g., linked to anantibiotic cassette and flanked by direct repeat sequences), or byligation of multiple copies and subsequent integration into thechromosome. In some embodiments, expression of the perhydrolase witheither the replicating plasmid or the integrated construct is monitoredusing the pNB activity assay (described herein) in an appropriateculture.

As with Bacillus, in some embodiments, expression of the perhydrolase inthe Gram-positive host Streptomyces is done using a replicating plasmid,while in other embodiments, expression of the perhydrolase isaccomplished via integration of the vector into the Streptomyceschromosome. Any promoter capable of being recognized in Streptomycesfinds use in driving transcription of the perhydrolase gene (e.g.,glucose isomerase promoter, A4 promoter). Replicating plasmids, eithershuttle vectors or Streptomyces only, also find use in the presentinvention for expression (e.g., pSECGT).

Structure of M. smegmatis Perhydrolase

The crystal structure of the M. smegmatis perhydrolase was determined to2.2 Angstroms. The structure confirmed findings with gel filtrationsizing columns, that indicated this enzyme is an octamer. The structureof the monomer places the enzyme in the class known as SGNH-hydrolases(See e.g., Molgaard et al., Structure 8: 373-383 [2000]). The activesite residues were identified as S11-D192-H195, based on homology,confirming the identification of the catalytic triad based on loss ofactivity in the S11A, D192A, and H195A mutations described above. FIG. 3provides schematics showing the structure of the M. smegmatisperhydrolase, as well as other serine hydrolases. As indicated, thisenzyme has a different structure than the enzymes shown here(chymotrypsin, subtilisin, and α/β hydrolase). Indeed, the structuralanalysis of the perhydrolases of the present invention indicates thatthis group of enzymes has a different form and active site than do theseother enzymes. A schematic diagram of the structure of the monomer isillustrated in FIG. 4. The structures of four other enzymes in theSGNH-hydrolase family have been solved, namely Aspergillus aculeatesrhamnogalucturonan acetylesterase (RGAE), Bos taurus platelet activatingfactor (PAF-AH(1b)a), Streptomyces scabies esterase (SsEst) and thethioesterase/Protease I/Phospholipase L₁ (TAP or Tes) from E. coli. Verylittle sequence or functional homology is present in these enzymes.Basically, the sequence identity is reserved for the residues involvedin the active site and those defining the family. While the overallfolding of the enzymes is similar (See e.g., Molgaard et al., supra[2000], for overlaying of structures), there are structural differences.For example, there is a loop covering the active site in SsEst, comparedto RGAE and TAP which have active sites that are surface-exposed. The M.smegmatis perhydrolase has an active site that is somewhat buried. Thebinding residues of the M. smegmatis perhydrolase were identified asCys7, Asp10, Ser11, Leu12, Thr13, Trp14, Trp16, Pro24, Thr25, Leu53,Ser54, Ala55, Thr64, Asp65, Arg67, Cys77, Thr91, Asn94, Asp95, Tyr99,Val125, Pro138, Leu140, Pro146, Pro148, Trp149, Phe150, Ile153, Phe154,Thr159, Thr186, Ile192, Ile194, and Phe196. These sites were derivedfrom direct observation and by modeling studies to model substratebinding to the enzyme, using methods known in the art.

As indicated above, the M. smegmatis perhydrolase was found to be anoctamer in the crystalline state. However, it is contemplated to beeither a hexamer or octamer in solution. The octamer is seen to be atetramer of dimers, two molecules are much more closely and extensivelyinteracting and these are termed the “act transferase” dimers. Severalof the conserved sites are found along this dimer interface. Forexample, residues Trp 14, Arg 27, Arg 56, His 81 and Pro 83, were foundto be conserved in natural isolates that have perhydrolase activity andare contemplated to be critical in forming the interface. In additionone other residue, Glu 51, which is conserved in all but one of thenatural isolates (and in that case it is a homologous enzyme) wasidentified.

One additional feature of interest in that in the natural isolatesshowing perhydrolase activity, all share an insertion of residues 69-81.This region forms a loop that is at the dimer interface. Without thisloop, it is believed that much of the dimer interface would be lost andit is likely that dimers and subsequent aggregation would not occur.Thus, there is a correlation of the insertion with the structuralaggregation particularly dimer formations and the appearance ofperhydrolase activity. However, it is not intended that the presentinvention be limited to any particular mechanisms.

Key residues were found to be associated with desired activity inselected homologs. Indeed, there are several conserved residues that arecontemplated to have importance for acyltransferase activity. Theseinclude Leu 6, Trp 14, Arg 27, Trp 34, Asp 62, Leu74, Leu 78 His 81,Pro83, Met 90, Lys 97, and Leu 114.

In additional analyses, the association of the perhydrolase withcarbamate was investigated. The native octamer was determined in spacegroup P4 with unit cell dimensions:

a=98.184 b=98.184 and c=230.119 α=90.00 β=90.00 γ=90.00, this crystaldiffracted to about 2.0 Å. The carbamate-inhibited crystal grew in thespace group P1 with unit cell dimensions a=67.754, b=80.096, andc=85.974 α=104.10°, β=112.10°, and γ=97.40° and these crystals diffractto a resolution exceeding 1.0 Å.

The carbamate was bound in a manner to exploit the interactions betweenthe keto oxygen of the carbamate and residues forming the oxyanion hole,the amide N atoms of Ser 11 and Ala 55 and Asn 94 ND2. The hydrophobicside chain extends along the hydrophobic surface of the binding site outinto the surface opening between pairs of dimers in the octamerstructure. The carbamate moiety direction highlights the pivotal role ofthe S54V mutation. The hydrophobic moiety passes adjacent to the sidechain of ser 54. Mutating the serine side to valine increased thehydrophobicity, and also served as a gatekeeper to prevent hydrophilicnucleophiles (e.g., water) for competing with desired deacylatingnucleophiles. The residues surrounding the carbamate moiety on the sameand neighboring molecules forming the extended entry are expected toinfluence the selection of the optimal de-acylating nucleophile. Thestructure showed that each monomer was inhibited with carbamatecovalently attached. Thus, all octamer active sites were found to beactive and functional. The side chain of carbamate resembles the leavinggroups of the substrates tested. Thus, the carbamate moiety indicatesthe access direction for substrate.

M. smegmatis Perhydrolase is an SGNH-Hydrolase

The perhydrolase of the present invention has certain components thatindicate it is in the SGNH-hydrolase family of enzymes. This family isdefined by having the four conserved amino acids SGN and H in fourblocks, similar to the blocks that describe the lipolytic family ofenzymes (See, Upton and Buckley, supra). In the case of the M. smegmatisperhydrolase, these correspond to S11, G52, N94 and H195 whichcorrespond to Blocks I II, III and V according to Upton and Buckley(Upton and Buckley, supra) and Molgaard et al. (Molgaard et al., supra).These amino acids are also conserved within the closest sequencehomologs of the perhydrolase.

As indicated herein, the sequences were aligned using the Alignmentprogram in Vector NTi (Informax, Invitrogen) In the following alignmentproviding a comparison of homolog sequences, the double underlineindicates the residues involved in the active site. AR: Agrobacteriumrhizogenes Q9KWA6; RR: Rhizobium rhizogenes NF006; SM: Sinorhizobiummeliloti RSM02162; MS: Mycobacterium smegmatis Act; MP: Mycobacteriumparafortuitum Phd partial sequence; PD: Prosthecobacter dejongeiiRVM04532. The amino acids within the blocks defining the SGNH-hydrolasefamily are indicated in bold letters.

                  Block I                                     Block II                    GDS                                         G AR(1)----------MAESRSILCFGDSLTWGWIPVPESSP TLRYPFEQRWTGAMAAALGDGYSIIEEGLSARTTSVED--PNRR(1)----------MAESRSILCFGDSLTWGWIPVPESSP TLRYPFEQRWTGAMAAALGDGYSIIEEGLSARTTSVED-PNRM(1)MTINSHSWRTLMVEKRSVLCFGDSLTWGWIPVKESSP TLRYPYEQRWTGAMAARLGDGYHIIEEGLSARTTSLDD-PNSM(1)-----------MVEKRSVLCFGDSRTWGWIPVKESSP TLRYPYEQRWTGAMAARLGDGYHIIEEGLSARTTSLDD-PNMS(1)-------------MAKRILCFGDSLTWGWVPVEDGAP TERFAPDVRWTGVLAQQLGADFEVIEEGLSARTTNIDD-PTMP-------------GIRRILSFGDSLTWGWIPVEEGVP TERFPRDVRWTGVLADLLGDRYEVIEEGLSARTTTAED-PAPD(1)--------------MKTILCFGDSNTWGYDPASMTAPFPRRHGPEVRWTGVLAKALGAGFRVIEEGQNGRTTVHED--PL                            Block III                            GxNDAR(67)DPRLNGSAYLPMALASHLPLDLVIILLGTNDTKSYFRRTPYEIANGMGKLAGQVLTSAGGIGTPYPAPKLLIVSPPPLAPRR(67)DPRLNGSAYLPMALASHLPLDLVIILLGTNDTKSYFRRTPYEIANGMGLKAGQVLTSAGGIGTPYPAPKLLIVSPPPLAPRM(78)DARLNGSTYLPMALASHLPLDLVIIMLGTNDTKSYFHRTPYEIANGMGKLVGQVLTCAGGVGTPYPAPKVLVVAPPPLAPSM(67)DARLNGSTYLPMALASHLPLDLVIIMLGTNDTKSYFHRTPYEIANGMGKLVGQVLTCAGGVGTPYPAPKVLVVAPPPLAPMS(65)DPRLNGASYLPSCLATHLPLDLVIIMLGTNDTKAYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMP(65)DPRLNGSQYLPSCLASHLPLDLVILMLATNDTKANFGRTPFDIATGMGVLATQVLTSAGGVGTSYPAPQVLIVAPPPLGEPD(65)NICRKGKDYLPACLESHKPLDLVILMLGTNDLKSTFNVPPGEIAAGAGVLGRMILAGDAGP-ENRPPQLLLMCPPKVRDL                                              Block V                                               DGIHF (SEQ ID NO: 14)AR(147)MPDPWFEGMFGGGYEKSLELAKQYKALANFLKVDFLDAGEFVKTDGCDGIHFSAETNITLGHAIAAKVEAIFSQEAKNAA(SEQ ID NO: 15) RR(147)MPDPWFEGMFGGGYEKSLELAKQYKALANFLKVDFLDAGEFVKTDGCDGIHFSAETNITLGHAIAAKVEAIFSQEAKNAA(SEQ ID NO: 16) RM(158)MPDPWFEGMFGGGYEKSKELSGLYKALADFMKVEFFAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF--------(SEQ ID NO: 17) SM(147)MPDPWFEGMFGGGYEKSKELSGLYKALADFMKVEFFAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF--------(SEQ ID NO: 18) MS(145)MPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEANNRDLGVALAEQVRSLL--------(SEQ ID NO: 19) MP 145)LPHPWFDLVFSGGREKTAELARVYSALASFMKVPFFDAGSVISTDMVDGI------------------------------(SEQ ID NO: 20) PD(144)SAMPDLDAKIPHGAARSAEFPRHYKAQAVALKCEYFNSQEIVETSPVDGIHLEASEHLKLGEALAEKVKVLLG-------

The primers used to identify homologs for each of the Blocks indicatedabove are provided below:

Block I (forward 5′-3) 1e: acggtcctgtgctttggngaytcnyt (SEQ ID NO: 21)1f: acggtcctgtgctttggngayagyyt (SEQ ID NO: 22) 1g:gcggtcctgttctwnggngaytcnyt (SEQ ID NO: 23) 1h:gcggtcctgttctwnggngayagyyt (SEQ ID NO: 24) 1i:gctcgaaccgtcctctgttttggngaytcnyt (SEQ ID NO: 25) 1j:gctcgaaccgtcctctgttttggngayagyyt (SEQ ID NO: 26) 1k:gctcgaaccgtcctctgtttnggngaytc (SEQ ID NO: 27) 1l:gctcgaaccgtcctctgttttggngaytcnytn (SEQ ID NO: 28) 1m:gctcgaaccgtcctctgttttggngaytcnytg (SEQ ID NO: 29) 1A:gccaagcgaattctgtgtttcggngaytcnyt (SEQ ID NO: 30) 1B:gccaagcgaattctgtgtttcggngayagyyt (SEQ ID NO: 31)Block III (reverse 5′-3) 3c: attccgcgcttcagrtcrttnvtncc (SEQ ID NO: 32)3d: attccgcgcttcagrtcrttnwgncc (SEQ ID NO: 33) 3e:attccgcgcttcagrtcrttnscncc (SEQ ID NO: 34) 3f:attccgcgcttcagrtcrttnrancc (SEQ ID NO: 35) 3k:attccgcgcttcagrtcrttnrtncc (SEQ ID NO: 36) 3l:attccgcgcttcagrtcrttnytncc (SEQ ID NO: 37) 3m:attccgcgcttcagrtcrttnsgncc (SEQ ID NO: 38) 3n:attccgcgcttcagrtcrttnwcncc (SEQ ID NO: 39) 3o:attccgcgcttcagrtcrttnyancc (SEQ ID NO: 40) 3p:attccgcgcttgrsrtcrttnrtncc (SEQ ID NO: 41) 3q:attccgcgcttgrsrtcrttnytncc (SEQ ID NO: 42) 3r:attccgcgcttgrsrtcrttnsgncc (SEQ ID NO: 43) 3s:attccgcgcttgrsrtcrttnwcnnn (SEQ ID NO: 44) 3t:attccgcgcttgrsrtcrttnyancc (SEQ ID NO: 45) 3A:gcgccggaagtaggccttggtrtcrttnvtncc (SEQ ID NO: 46) 3B:gcgccggaagtaggccttggtrtcrttnwgncc (SEQ ID NO: 47) 3C:gcgccggaagtaggccttggtrtcrttnscncc (SEQ ID NO: 48) 3D:gcgccggaagtaggccttggtrtcrttnrancc (SEQ ID NO: 49)Block III (forward 5′-3) 3g: cggaattatcatgctgggnabnaayga (SEQ ID NO: 50)3h: cggaattatcatgctgggncwnaayga (SEQ ID NO: 51) 3i:cggaattatcatgctgggngsnaayga (SEQ ID NO: 52) 3j:cggaattatcatgctgggntynaayga (SEQ ID NO: 53) 3u:ccggaattatcatgctnggnabnaayga (SEQ ID NO: 54) 3v:ccggaattatcatgctnggncwnaayga (SEQ ID NO: 55) 3w:ccggaattatcatgctnggngsnaayga (SEQ ID NO: 56) 3x:ccggaattatcatgctnggntynaayga (SEQ ID NO: 57) Block V (reverse 5′-3) 5c:acccttagcgtttggrtgnrtnccrtc (SEQ ID NO: 58) 5d:atccttagcgtttggrtgnavnccrtc (SEQ ID NO: 59) 5e:aatcttagccgtgrrrtgnrtnccrtc (SEQ ID NO: 60) 5f:aatcttagccgtgrrrtgnrcnccrtc (SEQ ID NO: 61) 5g:aatcttagccgtgrrrtgntrnccrtc (SEQ ID NO: 62) 5h:ccgctggtcctcatctggrtgnrtnccrtc (SEQ ID NO: 63) 5i:ccgctggtcctcatctggrtgnrcnccrtc (SEQ ID NO: 64) 5j:ccgctggtcctcatctggrtgntrnccrtc (SEQ ID NO: 65) 5k:ccgctggtcctcatcraartgnrtncc (SEQ ID NO: 66) 5A:cgattgttcgcctcgtgtgaartgnrtnccrtc (SEQ ID NO: 67) 5B:cgattgttcgcctcgtgtgaartgnrcnccrtc (SEQ ID NO: 68) 5C:cgattgttcgcctcgtgtgaartgntrnccrtc (SEQ ID NO: 69)

As described in greater detail herein, the sequence and structureresults are supported by the activity data that indicate theperhydrolase enzymes of the present invention differ from lipolyticenzymes known in the art.

Identification of Homologs

As well known in the art, proteins with a desired activity may beidentified in several ways, including but not limited to: 1) searchingavailable databases for proteins with sequence homology (30-100%); 2)screening environmental isolates for the desired activity; and 3)examining type strains from ATCC of the genus identified to haveactivities (e.g., Mycobacterium and Corynebacterium, as described hereinin particular embodiments).

By doing a standard protein-protein BLAST search, several homologs wereidentified from fully or partially sequenced genomes. From the knowngene sequence, several homologs were amplified by PCR from thechromosome of the parent organism and cloned into a pET expressionvector, essentially as described for the cloning of phd from M.smegmatis into pET16b. Homologues identified by this BLAST searchincluded: Agrobacterium rhizogenes Q9KWA6, A. rhizogenes Q9KWB1 A.tumefaciens Q8UFG4, A. tumefaciens Q8UAC0 (now AgrL, identical to 7-ACAarylesterase), A. tumefaciens Q9ZI09, A. tumefaciens (radiobacter) ACA,Prosthecobacter. dejongeii RVM04532, Rhizobium. loti Q98MY5, R. melilotiQ92XZ1, R. meliloti Q9EV56, R. rhizogenes NF006, R. rhizogenesNF00602875, R. solanacerarum Q8XQI0, Sinorhizobium meliloti RSM02162, S.meliloti RSM05666, Mesorhizobium loti RML000301, A. rhizogenes Q9KWA6,and A. rhizogenes Q9KWB1.

Based on these results, a homology tree of proteins with sequencehomology (20-80%) to M. smegmatis perhydrolase was generated. As shownin FIG. 2, an enzyme in the family of lipolytic enzymes described byUpton and Buckley (supra) is that of V. mimicus. This phylogenetic treewas generated using the alignment program in Vector NTi (Informax,Invitrogen). The green arrow indicates M. smegmatis perhydrolase, thered arrow indicates A. radiobacter 7-ACA arylesterase, the blue arrowindicates E. coli TAP, and the black arrow indicates A. aculeates RGAE.

As further indicated in FIG. 2, the perhydrolase is not closely relatedto this enzyme. The perhydrolase and its closest relatives,Prosthecobacter dejongeii RVM04532, R. rhizogenes NF006, A. rhizogenesQ9KWA6, R. meliloti Q92XZ1, S. meliloti RSM02162, A. rhizogenes Q9KWB1and R. rhizogenes NF00602875 come off their own branch (i.e., a branchthat is different from the 7-ACA arylesterase-like proteins and theRGAE/TAP-like proteins). However, it is contemplated that someadditional, more distantly related homologs will find use in the presentinvention due to perhydrolase activity or will serve as a suitablebackbone for modification to the desired perhydrolase activity.

In addition to the sequence and homology analysis, environmentalisolates were grown on a rich medium (N-MISO: g/l: glucose 10 g, yeastextract 10 g, KNO₃ 1.5, KH₂PO₄ 3.4 g, NaH₂PO₄.H₂0 3.4 g, Salt Solution C10 ml [Salt Solution C: g/l: MgSO₄.7H₂O 25, FeSO₄7H₂O 2.8, MnSO₄H₂O 1.7,NaCl 0.6, NaMoSO₄.2H₂O, ZnSO₄.7H₂O 0.06, in 0.1N HCl]), assayed andthose positive for the transesterification reaction were purified asdescribed in the Examples. This is one of the screening methods that canbe used to identify perhydrolase. These data show that the presentinvention finds use in identification of additional enzymes with thedesired perhydrolase activity.

Additional Investigations of Homologues

In addition to the above analyses, an enzyme library of novel“GDSL-type” esterases which are homologous to the prototype M. smegmatisperhydrolase was created. In order to identify new “GDSL”-typeesterases, a sequence homology based screening procedure was establishedand used to screen libraries set up from complex metagenomic DNA (atBRAIN).

An enzyme library comprising 19 “GDSL”-type esterases (See, below) wasdeveloped. The sequences in this library were:

S248_M2bB11 (DNA) (SEQ ID NO: 70)ATGTTCGCGCTTTGCACGGCCGCGTCAGCGGCCCCCGATCGCACCGTCGTCTTTTTTGGGGACAGCCTGACCGCGGGGTACGGCCTCGATGACCCGCAGACCCAGTCCTACCCGGCCAGGATCCAGGAGAAGGTCGACGCCGCGGGCCTGCGCTGGAAGGTCGTGAATGCCGGCCTCTCGGGCGAGACGAGCGCCGGCGGCCTGCGGCGGGTCGACTGGGTGCTCGGCCAGCACATCGACGCCTTTGTCCTGGCGCTTGGCGCCAACGATGGCCTGCGGGGGATCGACCCCCAGGTCACGAGGGCCAATCTCCAGGAGATCATCAACCGGGTCCGCTCCCGGTGGCCCCGCGCGGCGATCGTCATCGCCGGGATGAAAATGCCCCAGAGCATGGGACAGGACTACGCCGCGAATTTTGACCGGATCTTCCCCGGTCTCGCCGCGAGGAATTCGGCCACGCTCATCCCCTTTCTATTAGAAGGGGTCGCCGCCCATCCTAGCCTCAACCAAGGCGACGGCATCCACCCGACGGCCGCCGGGGACGCACTCGTTGCAGGGACCGTGTGGACGTACCTGCTTCCGATCCTGCGGTCAGCACAC TAAS248_M2bB11 (Amino Acid) (SEQ ID NO: 71)MFALCTAASAAPDRTVVFFGDSLTAGYGLDDPQTQSYPARIQEKVDAAGLRWKVVNAGLSGETSAGGLRRVDWVLGQHIDAFVLALGANDGLRGIDPQVTRANLQEIINRVRSRWPRAAIVIAGMKMPQSMGQDYAANFDRIFPGLAARNSATLIPFLLEGVAAHPSLNQGDGIHPTAAGDALVAGTVWTYLLPILRSAH S248_M40cD4 (DNA)(SEQ ID NO: 72) ATGCGCTTTGCTAAGCTCACTGCCGTCATCTTTGCCCTGATAGTCTTGCACAGCCCCCTTGCCGCCGCCGCGCCGCCCACCGTGATGGTGTTTGGCGACAGTCTGACCGCCGGGTTGGGATTGCCGGCCGATGCTGCATTTCCGGCGCAGCTCCAGGCAAAGCTGCACGATATGGGTATCCTGCAGAAATCGCCGCGCGCGCCACCTCGGGGCAAACGACGGCCGGCGGGTTGGCGAGCCTTGCGGATGCGCTGGCCGCAAAGCCGGATTTGGTGATCCTCGAACTCGGCGCCAATGACATGCTGCGCGCGGTCGATCCGGCCAGCGTGCGCGCCAATCTCGATGCAATGATGACGAAAATCCAGGCGAGCGGCGCTAAACTGCTGCTGACCGGAATGCAGGCGGCGCCCAATTGGGGCGAGGACTATAAGCACGATTTCGACCGCCTTTATCCCGAGCTTGCGAAGGCGCACGGGGTGACGCTTTATCCATTCTTTCTTGATGGGGTGGCGCTGGACCCGGCGCTGAACCAGGCGGATGGAATGCACCCGAACGCCAAGGGGGTCGCCGTGATCGTCGACCGTATCGCGCCCGTCGTCGCCAAGATGCTGAGAGGCCAGTCATAA S248_M40cD4 (Amino Acid) (SEQ ID NO: 73)MRFAKLTAVIFALIVLHSPLAAAAPPTVMVFGDSLTAGLGLPADAAFPAQLQAKLHDMGIPAEIAARATSGQTTAGGLASLADALAAKPDLVILELGANDMLRAVDPASVRANLDAMMTKIQASGAKLLLTGMQAAPNWGEDYKHDFDRLYPELAKAHGVTLYPFFLDGVALDPALNQADGMHPNAKGVAVIVDRIAPVV AKMLRGQSS248_M44aA5 (DNA) (SEQ ID NO: 74)ATGATCGCATGGCTTACCGGATGCGGCAGCGCAAAGACGCAACCGCAGCCCGCAAGTTCCATCCCGCCATCCAGTATTCCAGCAACCGCAAAACCTGCGACAACGGATATCAGACCGATCATCGTTGCTTTCGGCGACAGCCTGACTGCAGGATACGGCGTCAGTAGTGAACAAAGCTATCCGGCCAATCTTCAACGCGATCTGGATGCGCGTGGATATCATGCCCACGTCATCAACGAAGGCATCAGCGGCAACACATCGAAAGACGGCGTTCTCAGGGCCCAGGCGATTGCGGCACTCCATCCGGCTGTCGTCATCGTTGCCTTCGGCGGCAACGACGGTCTGCGTGGCCTCCCCATCGGAGACACGGAAATGAATCTGGCAACGATCATCTCAACCATGCAGCATGCCCATGCCAAGGTAATTTTAGGCGGAATTACTTTGCCTCCCAACTATGGCAGCGAATACATCGCCAAATTCAATGCGATCTATAAAAAGCAGGCAGCCGCGTATCATGTGCCCCTGCTGCCCTTCATGCTGAAGGGGGTGTATGGCGTGCCCGGTTCCATGCAGAGCGACGGCATCCATCCGACCGCCAAGGGCTGCCAGCAAGTGGCCAGAAACTTCCTGCCCTTGTTATTGCCGCTCCTGCACAAATCAGGGAAGAAATCCATGGAGTCGAAAGCATTGTCTCGACGTC ATTAAS248_M44aA5 (Amino Acid) (SEQ ID NO: 75)MIAWLTGCGSAKTQPQPASSIPPSSIPATAKPATTDIRPIIVAFGDSLTAGYGVSSEQSYPANLQRDLDARGYHAHVINEGISGNTSKDGVLRAQAIAALHPAVVIVAFGGNDGLRGLPIGDTEMNLATIISTMQHAHAKVILGGITLPPNYGSEYIAKFNAIYKKQAAAYHVPLLPFMLKGVYGVPGSMQSDGIHPTAKGCQQVARNFLPLLLPLLHKSGKKSMESKALSRRH S261_M2aA12 (DNA) (SEQ ID NO: 76)ATGAAAAACATCCTTGCATTTGGCGACAGTCTGACCTGGGGTTTTGTGGCCGGACAGGATGCGCGCCATCCGTTTGAAACCCGCTGGCCAAACGCATTGGCGGCCGGCCTTGGGGGCAAAGCCCGCGTAATTGAAGAGGGTCAGAACGGCCGCACTACGGTGTTCGACGATGCCGCCACCTTCGAATCTCGAAATGGCTCGGTGGCATTGCCGCTGCTACTGATCAGCCACCAGCCGTTGGACCTGGTAATCATCATGCTCGGCACCAATGACATCAAGTTTGCCGCCCGCTGCCGCGCCTTTGATGCTTCAATGGGCATGGAACGGCTGATCCAGATCGTCAGAAGTGCCAACTACATGAAGGGCTACAAGATACCTGAAATCCTCATCATATCGCCGCCCAGCCTCGTGCCGACGCAGGATGAATGGTTCAACGACCTCTGGGGCCATGCCATCGCCGAGTCAAAACTCTTCGCCAAGCACTACAAGCGCGTGGCCGAAGAACTGAAAGTGCATTTCTTTGATGCAGGCACGGTGGCCGTCGCCGACAAGACCGACGGCGGACATCTCGATGCTGTGAATACTAAAGCCATTGGCGTCGCATTGGTGCCGGTGGTGAAATCAATACTCGCTCTCTAA S261_M2aA12 (Amino Acid)(SEQ ID NO: 77) MKNILAFGDSLTWGFVAGQDARHPFETRWPNALAAGLGGKARVIEEGQNGRTTVFDDAATFESRNGSVALPLLLISHQPLDLVIIMLGTNDIKFAARCRAFDASMGMERLIQIVRSANYMKGYKIPEILIISPPSLVPTQDEWFNDLWGHAIAESKLFAKHYKRVAEELKVHFFDAGTVAVADKTDGGHLDAVNTKAIGV ALVPVVKSILALS279_M70aE8 (DNA) (SEQ ID NO: 78)ATGCCGAAAATAGCCAAACTCGCGCCGTCGGATGTGATCGTAGCTTTCGGCGACAGTCTGACGTTCGGCACCGGCGCAACGGAAGCGGAGAGTTATCCCATCGTGCTCGCACAATTGATCGGTCGCACCGTGGTGCGCGCGGGTGTGCCGGGTGAGGTAACCGAAGGCGGGCTTGCGCGCCTGACCGACGTTATCGAAGAACACAAGCCGAAGCTGATTATTGTTTGCCTGGGCGGCAACGACATGCTGCGCAAGGTCCAGGAAGACCAGACCCGCGCCAATTTGCGCGCCATTATTAAAACCATCAAGGCGCAAGGCATCGCCGTGGTACTGGTCGGTGTGCCGAAGCCCGCGCTGGTGACCAGTGCGCCGCCGTTCTACGAGGAGATCGCCAAAGAGTTCGGTATCCCTTACGAAGGCAAGATTGTTACCGACGTGTTGTACCAACGCGATCAGAAATCCGATTCCATACATCCCAATGCCAAAGGCTATCGGCGCATGGCCGAAGCGATAGCCACGCTGCTGAAAAAATCCGGAGCCATTTAA S279:M70aE8 (Amino Acid)(SEQ ID NO: 79) MPKIAKLAPSDVIVAFGDSLTFGTGATEAESYPIVLAQLIGRTVVRAGVPGEVTEGGLARLTDVIEEHKPKLIIVCLGGNDMLRKVQEDQTRANLRAIIKTIKAQGIAVVLVGVPKPALVTSAPPEYEEIAKEFGIPYEGKIVTDVLYQRDQKSDSIHPNAKGYRRMAEAIATLLKKSGAI S279_M75bA2 (DNA) (SEQ ID NO: 80)ATGGAACGGACCGGCCGCGCTGGCGATCGGTGTCGGCGTGGGGCTGGCGAGCCTGAGCCCGGTCGCGCTGGCGACGCCGCCGCGGGGCACCGTGCCGGTGTTCACCCGATCGGGGACAGCCTGACGGACGAGTATTTTGAGCCGTTCTTCCAGTGGGGGTTCTGCGGGAAGTCGTGGGCCGAGATTTTGGTGGAGACGGGGCGGGCGAGCATGGGCCCGACGGCGCAGCAGGCGGGGATCAGCGAGCCGGAGGGATGGTCGGATCCGCGGAACACGGGGTATCAGCACAACTGGGCGCGGTACTCGTGGAGCTCCTCAGACGCGCTGACCGAGGAGTCGCCGGGGGCGACGCTGAGCGTGCTGCTTGGGGCGGAGTACGCGGTGGTGTTCATTGGGACCAACGACTTCAATCCGTCGTGGCCGGCGTATCAGAGCGTGTATCTGAGCCAGTGGAGCGACGAGCAGATCGACACGTACGTGAACGGGGTGGTGCAGAACATCGCGCAGATGGTGGACTCGCTGAAGTCGGTCGGGGCGAAGGTGGTGCTTGCGCCGCCGGTGGATTTTCAGTTCGCGGGGTTCCTGCGGAACTCATGCCCGGATCCGATGCTGCGCGAGCAGGCGGGTATTCTGACACGGAAGTGCCACGACCGGGTGCGGTCGATGGCGCGGCAGAAGCACGTGGTGTTCGTGGACATGTGGCGGCTGAACCGCGATTTGTTCGGCAACGGGTTCGCGATCAGCTACGGCCTTCGGAACACGGTGCGCGTGGGGGACTCGGAGATCGGGCTGCAACTGGCCGGGCTGACGGGATCGGCGGGGCTGGTTCCGGACGGGATCCATCCGCAGCGGGTGGTGCAGGGGATCTGGGCGAATGCGTTCATCGTGGGTCTGAACGCGCATGGGGCGAACATCGCGCCCATCGGCGAGGCGGAGATGTGCGCGATGGGGGGGGTCGTGTACGGGGGAACGGACACGCTGGCGAACTTCCTGCCGCCGGTCGCGGGCTACGTGGAGGACTTCCGCAACGCGGGGGACTTCGTGTGCACGGCGGACTTCAACCATGACCTTGGCGTGACGCCGACGGACATCTTCGCGTTCATCAACGCGTGGTTCATGAATGATCCCTCGGCGCGGATGAGCAACCCGGAGCACACGCAGATCGAGGACATCTTCGTGTTTCTGAATCTGTGGCTGGTG GGGTGCTAAS279_M75bA2 (Amino Acid) (SEQ ID NO: 81)MERTGRAGDRCRRGAGEPEPGRAGDAAAGHRAGVHPIGDSLTDEYFEPFFQWGFCGKSWAEILVETGRASMGPTAQQAGISEPEGWSDPRNTGYQHNWARYSWSSSDALTEESPGATLSVLLGAEYAVVFIGTNDFNPSWPAYQSVYLSQWSDEQIDTYVNGVVQNIAQMVDSLKSVGAKVVLAPPVDFQFAGFLRNSCPDPMLREQAGILTRKCHDRVRSMARQKHVVFVDMWRLNRDLFGNGFAISYGLRNTVRVGDSEIGLQLAGLTGSAGLVPDGIHPQRVVQGIWANAFIVGLNAHGANIAPIGEAEMCAMGGVVYGGTDTLANFLPPVAGYVEDFRNAGDFVCTADFNHDLGVTPTDIFAFINAWFMNDPSARMSNPEHTQIEDIFVFLNLWLV GC M091_M4aE11 (DNA)(SEQ ID NO: 82) ATGAAGACCATTCTCGCCTATGGCGACAGCCTGACCTATGGGGCCAACCCGATCCCGGGCGGGCCGCGGCATGCCTATGAGGATCGCTGGCCCACGGCGCTGGAGCAGGGGCTGGGCGGCAAGGCGCGGGTGATTGCCGAGGGGCTGGGTGGTCGCACCACGGTGCATGACGACTGGTTTGCGAATGCGGACAGGAACGGTGCGCGGGTGCTGCCGACGCTGCTCGAGAGCCATTCGCCGCTCGACCTGATCGTCATCATGCTCGGCACCAACGACATCAAGCCGCATCACGGGCGGACGGCCGGCGAGGCCGGGCGGGGCATGGCGCGGCTGGTGCAGATCATCCGCGGGCACTATGCCGGCCGCATGCAGGACGAGCCGCAGATCATCCTCGTGTCGCCGCCGCCGATCATCCTCGGCGACTGGGCGGACATGATGGACCATTTCGGCCCGCACGAAGCGATCGCCACCTCGGTGGATTTCGCTCGCGAGTACAAGAAGCGGGCCGACGAGCAGAAGGTGCATTTCTTCGACGCCGGCACGGTGGCGACGACCAGCAAGGCCGATGGCATCCACCTCGACCCGGCCAATACGCGCGCCATCGGGGCAGGGCTGGTGCCGCTGGTGAAGCAGGTGCTCGGCCTGTAAM091_M4aE11 (Amino Acid) (SEQ ID NO: 83)MKTILAYGDSLTYGANPIPGGPRHAYEDRWPTALEQGLGGKARVIAEGLGGRTTVHDDWFANADRNGARVLPTLLESHSPLDLIVIMLGTNDIKPHHGRTAGEAGRGMARLVQIIRGHYAGRMQDEPQIILVSPPPIILGDWADMMDHFGPHEAIATSVDFAREYKKRADEQKVHFFDAGTVATTSKADGIHLDPANTRA IGAGLVPLVKQVLGLEst105 (DNA) (SEQ ID NO: 84)ATGCGCACGCTTCACCGAAGCCTGCTCGCAAGCGCGGCCGCGCTTTTTCTAGCGGCATCCGGCAACGCAACGGCGCAGTTCTCGAACGTCTATTTCTTCGGCGACAGCCTGACCGACGCGGGTTCCTTCAAGCCTGTGCTGCCTCCTGGTACAGGATTATTCACGACGAATCCCGGCCCGGTATGGCCGCAGGTATTCGGGGCGAACTACGGCGTCGCGGTGACGCCCGCAAACCAGGGTGGGACCGATTATGCGCAGGGTGGCGCGCGCGTGACGAGCCTGCCTGGCGTTCCGACGTCGCAGCCGACCGGCAGCGCGGTACCGATCGCTACGCAGATTTCGCAGTTCCTCGGCTCGGGTCCGGCGGATCCGAACGCATTCTATTCGGTGTGGGGCGGCGCGAACGACATCTTTTTCCAGCTGGGGTTGGCGCAGGCGGGCATGGCGACGCCGGCGCAGGTCCAGTCGGCCGTCGGCTTGGCCGCGGTCCAGCTGGCGCAGGCAACTGCGGCGCTCAACGCCAGCGGCGCGCGATTCATCACGGTTATCAACGTGCCGGACATCGGTAAAACGCCGTTCGGCGTCGGCTCCGGTCAAGGAGCGCAGATCACCGCTCTGTCGTCTTTCTTCAACAGCACGCTGTTCGGCGCGCTCGACGCCACGGGCATCCAGACGATGCGCGTGAACGGGTTCGCGGTGCTGAACGAGGTGGTCGCGGACCCGGCGGCTTATGGCTTCGCGAATGCATCAACGCCAGCGTGCGGGGCCACGCCATCGCTCGTCTGCACGTCGGCGAACTTCGTCACGCCCTTGGCCGCGCAGACCTTCCTCTTCGCAGACGGCGTTCACCCCACCACGGCCGGGCACGCCCTCATCGCCCAAGCGGTCCAGGCGATGATCACCGGTCCCCAACAGATGGCGGCGTTGGGCGACGCCCCGCTCGCCGTCGAGCAGGCCAACTTCCGCGCGCTCGACAACCGCATGTGGTCGAGCCTCAATGCGCCGCGCAGCCCGGGCAAGCTCCAGGGTTGGGCGGCCTACGACTACAGCCACACGGACCTGCAGGCGGGACCGACCAATGGCAGCGGACACATGAACACCGTTGCGGTCGGGGTCGACATGAAAGTCTCCGATCATATGCTCGCCGGCGCGATGTTCGGCTATACCAACACCAAGGGCGACTTCGGCGGCCCCGGCGGCGGATACACACTGAAGCAGCCTGTGGGCACTGCCTATGCGGGTTACGGCGTGGGCCCTTGGTATGTCGGCGCGACGCTCGGCACAGGTGGCCTCGACTACTCGGACGTCACGCGCGCCATCCCGCTTGGCTTGGCGGTTCGCACCGAGAGCGCCGAGGCCCGAGGCTACGAGTTCACGGGCCGGATCCTCGGCGGCTACTGGTTCACGATGCGCGACCTGATGCACGGGCCGTACGCGCGTCTCGCGTGGACGAAGGCCGTCGTCAAGCGGTTTTCCGAGGAGAGCACCGACAGCACGGCGTTGAACTACGACAGGCAGGAGCGCAAGCAACTGCTGTGGAGCCTCGGATGGCAACTCGCCGGCAACGTCGGCAGCATCCGTCCCTACGCGCGGGCGACCTGGGAGATCGACTCCAAGGATCAGGACCGCAGCGTTGGCGCATCGTCGGTCACGCTGGGCGGCTTTTACAGTGTTCCGGTCGCGAAGCCGGACAATAGCTATGCGCTCTTCAGCCTCGGCGCGAGTACCGAGCTCGGGAGCGTCACCGGGTTTGTCGCGGGCTCGGCCACCGCAGGCCGGGCGGATGCCAACTATTGGGCGGTCACGGTCGGCCTGCGGATGCCGTTGTAG Est105 (Amino Acid) (SEQ ID NO: 85)MRTLHRSLLASAAALFLAASGNATAQFSNVYFFGDSLTDAGSFKPVLPPGTGLFTTNPGPVWPQVFGANYGVAVTPANQGGTDYAQGGARVTSLPGVPTSQPTGSAVPIATQISQFLGSGPADPNAFYSVWGGANDIFFQLGLAQAGMATPAQVQSAVGLAAVQLAQATAALNASGARFITVINVPDIGKTPFGVGSGQGAQITALSSFFNSTLFGALDATGIQTMRVNGFAVLNEVVADPAAYGFANASTPACGATPSLVCTSANFVTPLAAQTFLFADGVHPTTAGHALIAQAVQAMITGPQQMAALGDAPLAVEQANFRALDNRMWSSLNAPRSPGKLQGWAAYDYSHTDLQAGPTNGSGHMNTVAVGVDMKVSDHMLAGAMFGYTNTKGDFGGPGGGYTLKQPVGTAYAGYGVGPWYVGATLGTGGLDYSDVTRAIPLGLAVRTESAEARGYEFTGRILGGYWFTMRDLMHGPYARLAWTKAVVKRFSEESTDSTALNYDRQERKQLLWSLGWQLAGNVGSIRPYARATWEIDSKDQDRSVGASSVTLGGFYSVPVAKPDNSYALFSLGASTELGSVTGFVAGSATAGRADANYWA VTVGLRMPLEst114 (DNA) (SEQ ID NO: 86)ATGGGGCGATCGAGAGTTCTGAAGGCTGTTTTCCTGGTGGCGTGCCTTGTGGGTCGGCTCGCGGCGCATGCCGAGGCGTCGCCCATCGTGGTCTACGGCGATAGCCTCTCTGACAACGGCAATCTGTTTGCGCTCACCGGCGGTGTCGCGCCGCCCTCGCCGCCGTACTTCAACGGACGGTTTTCTAATGGCCCGGTGGCCGTGGAGTATCTCGCGGCCGCGCTGGGATCTCCGCTGATCGATTTCGCGGTCGGCGGGGCGACGACCGGCCTCGGCGTCAACGGCGATCCCGGTGGTTCGCCGACGAGTCTCGGCGCGGCGGGATTGCCGGGGCTTCAGACGACATTCGCCGCCACGCAAGGCACGCTGGGTCCGTACGTTGGTGGTCTCTTCGTGGTGTGGGCGGGTCCGAACGACTTCTTGTCGCCCTCGCCGCTTGACACGAACGCTTTTCAGATTGCGAACCGGGCCGTGTCCAACATCCTCGGCGTGGTGGCATCACTTCAGGCACTCGGCGTCGAGCGCATCCTCGTCCCCGGCATGCCCGATCTCGGTCTGACGCCCGCTCTTCAGCCCATCGCAGGCGCAGCCACCGCGTTCACCGATTTGTTCAACTCGATGCTGCGCGCGGGCTTGCCGAACGACGTGCTGTACCTGGACACGGCGACAATCTTCCGATCGATCGTGGCAGACCCTGGGGCCTACGGCTTGACCAACGTGACCACGCCGTGCCTGATTGGTGCGACCGTCTGCGCGAATCCGGATCAGTACCTGTTCTGGGATGGTATTCATCCTACGACGGCGGGGCACGCGATCTTGGGCAATGCCCTCGTCGCCCAGGCAGTCCCCGAGCCCGCGACCATGGTGCTCGTGCTGACGGGTCTGTCCATGCACGTGATT GCGCGCCGGCGGCGGGCGTAAEst114 (Amino Acid) (SEQ ID NO: 87)MGRSRVLKAVFLVACLVGRLAAHAEASPIVVYGDSLSDNGNLFALTGGVAPPSPPYFNGRFSNGPVAVEYLAAALGSPLIDFAVGGATTGLGVNGDPGGSPTSLGAAGLPGLQTTFAATQGTLGPYVGGLFVVWAGPNDFLSPSPLDTNAFQIANRAVSNILGVVASLQALGVERILVPGMPDLGLTPALQPIAGAATAFTDLFNSMLRAGLPNDVLYLDTATIFRSIVADPGAYGLTNVTTPCLIGATVCANPDQYLFWDGIHPTTAGHAILGNALVAQAVPEPATMVLVLTGLSMHVI ARRRRASinorhizobium meliloti SmeI (SMa1993) (DNA) (SEQ ID NO: 88)ATGACAATCAACAGCCATTCATGGAGGACGTTAATGGTGGAAAAGCGCTCAGTACTGTGCTTTGGGGATTCGCTGACATGGGGCTGGATTCCGGTGAAGGGATCCTCACCGACCTTGCGCTATCCCTATGAACAACGGTGGACCGGCGCAATGGCCGCGAGGCTTGGCGACGGTTACCACATCATCGAAGAGGGGCTGAGCGCCCGCACCACCAGCCTCGACGACCCCAACGACGCGCGGCTCAACGGCAGCACCTACCTGCCCATGGCACTCGCCAGCCACCTCCCACTCGACCTCGTCATCATCATGCTGGGCACGAACGACACGAAATCCTATTTCCACCGCACGCCTTACGAGATCGCCAACGGCATGGGCAAGCTAGTCGGCCAGGTGCTGACCTGCGCCGGTGGCGTCGGCACGCCATATCCCGCGCCGAAGGTGCTTGTCGTCGCTCCGCCGCCGCTCGCGCCGATGCCCGACCCGTGGTTCGAAGGCATGTTCGGCGGCGGCTACGAGAAGTCGAAGGAACTCTCCGGCCTCTACAAGGCGCTTGCCGATTTCATGAAGGTCGAGTTTTTCGCCGCCGGTGATTGCATTTCCACCGATGGGATCGACGGCATTCACCTCTCGGCGGAAACCAACATCAGACTCGGGCACGCGATCGCGGACAAAGTTGCGGCGTTGTTCSinorhizobium meliloti SmeI (SMa1993) (Amino Acid) (SEQ ID NO: 89)MTINSHSWRTLMVEKRSVLCFGDSLTWGWIPVKGSSPTLRYPYEQRWTGAMAARLGDGYHIIEEGLSARTTSLDDPNDARLNGSTYLPMALASHLPLDLVIIMLGTNDTKSYFHRTPYEIANGMGKLVGQVLTCAGGVGTPYPAPKVLVVAPPPLAPMPDPWFEGMFGGGYEKSKELSGLYKALADFMKVEFFAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALFSinorhizobium meliloti SmeII (Q92XZ1) (DNA) (SEQ ID NO: 90)ATGGAGGAGACAGTGGCACGGACCGTTCTATGCTTCGGAGATTCCAACACTCACGGCCAGGTACCTGGCCGCGGACCGCTTGATCGCTACCGACGCGAACAGCGCTGGGGCGGTGTTCTGCAAGGCCTGCTCGGCCCGAACTGGCAGGTTATCGAAGAAGGCCTGAGCGGACGCACGACCGTGCATGACGATCCGATCGAAGGTTCGCTCAAGAACGGCCGGACCTATCTGCGCCCCTGTCTGCAGAGCCATGCACCACTCGACCTTATCATCATTATGCTCGGCACCAATGACCTGAAGCGGCGCTTCAACATGCCACCGTCCGAGGTCGCAATGGGCATCGGCTGTCTCGTGCACGATATCCGAGAACTCTCGCCCGGCCGGACCGGCAACGATCCCGAAATCATGATCGTCGCCCCGCCGCCGATGCTGGAAGATCTCAAGGAATGGGAGTCGATTTTCTCAGGCGCACAGGAAAAATCTCGCAAGCTGGCGCTGGAGTTCGAGATAATGGCGGATTCTCTGGAGGCGCATTTCTTCGACGCCGGTACGGTCTGCCAGTGTTCGCCGGCCGATGGCTTCCACATCGACGAGGATGCCCACCGCCTGCTCGGCGAGGCTCTCGCCCAGGAAGTGCTGGCGATCGGGTG GCCCGATGCGTAASinorhizobium meliloti SmeII (Q92XZ1) (Amino Acid) (SEQ ID NO: 91)MEETVARTVLCFGDSNTHGQVPGRGPLDRYRREQRWGGVLQGLLGPNWQVIEEGLSGRTTVHDDPIEGSLKNGRTYLRPCLQSHAPLDLIIIMLGTNDLKRRFNMPPSEVAMGIGCLVHDIRELSPGRTGNDPEIMIVAPPPMLEDLKEWESIFSGAQEKSRKLALEFEIMADSLEAHFFDAGTVCQCSPADGFHIDEDA HRLLGEALAQEVLAIGWPDASinorhizobium meliloti SmeIII (Q9EV56) (DNA) (SEQ ID NO: 92)ATGAAGACAGTCCTTTGCTACGGTGACAGTCTGACCTGGGGATACGATGCAACCGGTTCCGGCCGGCATGCGCTGGAGGACCGTTGGCCGAGCGTGCTGCAGAAGGCGCTCGGTTCGGACGCGCATGTCATCGCCGAAGGGCTGAACGGGCGGACGACCGCCTATGACGACCATCTCGCCGATTGCGACCGGAACGGCGCGCGTGTCCTCCCGACGGTCCTGCACACCCACGCGCCACTCGATCTCATCGTGTTCATGCTCGGCTCGAACGACATGAAGCCGATCATTCACGGCACCGCTTTCGGCGCGGTGAAGGGCATCGAGCGCCTCGTCAATCTGGTGCGCAGGCACGACTGGCCGACGGAAACGGAGGAGGGGCCCGAGATTCTCATCGTCTCGCCGCCGCCGCTCTGCGAGACGGCCAACAGCGCCTTTGCCGCCATGTTCGCGGGCGGGGTCGAGCAATCCGCAATGCTGGCGCCGCTTTATCGCGATCTCGCCGACGAGCTCGACTGCGGCTTCTTCGACGGCGGATCGGTGGCCAGGACGACGCCGATCGACGGTGTCCACCTCGACGCGGAGAACACCCGGGCGGTCGGCAGAGGGTTGGAGCCTGTCGTGCGGATGATGCTCGGGCTTTAASinorhizobium meliloti SmeIII (Q9EV56) (Amino Acid) (SEQ ID NO: 93)MKTVLCYGDSLTWGYDATGSGRHALEDRWPSVLQKALGSDAHVIAEGLNGRTTAYDDHLADCDRNGARVLPTVLHTHAPLDLIVFMLGSNDMKPIIHGTAFGAVKGIERLVNLVRRHDWPTETEEGPEILIVSPPPLCETANSAFAAMFAGGVEQSAMLAPLYRDLADELDCGFFDGGSVARTTPIDGVHLDAENTRAVG RGLEPVVRMMLGLAgrobacterium tumefaciens Atu III (AAD02335) (DNA) (SEQ ID NO: 94)ATGGTGAAGTCGGTCCTCTGCTTTGGCGATTCCCTCACCTGGGGATCAAATGCGGAAACGGGTGGCCGGCACAGCCATGACGATCTTTGGCCGAGCGTCTTGCAGAAGGCGCTCGGTCCTGACGTGCATGTGATTCACGAAGGTCTGGGTGGTCGCACCACCGCCTATGACGACAACACCGCCGATTGCGACCGCAACGGCGCGCGGGTTCTTCCGACGTTGTTGCACAGCCATGCGCCGCTGGATCTGGTGATTGTCATGCTCGGGACCAACGACCTGAAGCCGTCAATCCATGGATCGGCGATCGTTGCCATGAAGGGTGTCGAAAGGCTGGTGAAGCTCACGCGCAACCACATCTGGCAGGTGCCGGACTGGGAGGCGCCTGACGTGCTGATCGTCGCACCGCCGCAGCTGTGTGAAACGGCCAATCCGTTCATGGGCGCGATCTTTCGTGATGCGATCGATGAATCGGCGATGCTGGCGTCCGTTTACCGGGACCTTGCCGACGAGCTTGATTGCGGCTTTTTCGATGCGGGTTCCGTCGCCCGAACGACGCCGGTGGATGGCGTTCATCTCGATGCTGAAAATACGCGGGCCATCGGGCGGGGGCTGGAGCCCGTCGTTCGCATGATGCTCGGACTTTAAAgrobacterium tumefaciens Atu III (AAD02335) (Amino Acid)(SEQ ID NO: 95) MVKSVLCFGDSLTWGSNAETGGRHSHDDLWPSVLQKALGPDVHVIHEGLGGRTTAYDDNTADCDRNGARVLPTLLHSHAPLDLVIVMLGTNDLKPSIHGSAIVAMKGVERLVKLTRNHIWQVPDWEAPDVLIVAPPQLCETANPFMGAIFRDAIDESAMLASVYRDLADELDCGFFDAGSVARTTPVDGVHLDAENTRAI GRGLEPVVRMMLGLMesorhizobium loti Mlo I (Q98MY5) (DNA) (SEQ ID NO: 96)ATGAAGACGGTGCTTTGCTACGGCGACTCGCTGACCTGGGGCTACAATGCCGAAGGCGGCCGCCATGCGCTGGAAGACCGCTGGCCGAGCGTGCTGCAAGCAGCGTTAGGCGCCGGCGTGCAAGTGATTGCCGATGGCCTCAACGGCCGCACCACGGCCTTCGACGATCATCTGGCCGGTGCTGATCGCAACGGCGCCAGGCTGCTGCCGACGGTCCTGACGACGCACGCGCCGATCGACCTGATCATCTTCATGCTCGGCGCCAACGACATGAAGCCTTGGATCCACGGCAATCCGGTCGCAGCCAAGCAAGGCATCCAGCGGTTGATCGACATCGTGCGTGGTCACGACTACCCGTTCGACTGGCCGGCGCCGCAGATCCTGATCGTCGCGCCGCCTGTAGTCAGCCGCACCGAAAATGCCGACTTCAAGGAAATGTTCGCCGGTGGCGATGACGCCTCGAAGTTTTTGGCACCGCAATATGCCGCGCTCGCCGACGAAGCCGGCTGTGGCTTCTTCGACGCCGGCAGCGTGGCCCAAACCACACCGCTCGATGGCGTTCACCTCGATGCCGAAAACACGCGAGAAATCGGCAAGGCGCTGACGCCGATCGTGCGCGTCATGCTGGAATTGTAA Mesorhizobium loti Mlo I (Q98MY5)(Amino Acid) (SEQ ID NO: 97)MKTVLCYGDSLTWGYNAEGGRHALEDRWPSVLQAALGAGVQVIADGLNGRTTAFDDHLAGADRNGARLLPTVLTTHAPIDLIIFMLGANDMKPWIHGNPVAAKQGIQRLIDIVRGHDYPFDWPAPQILIVAPPVVSRTENADFKEMFAGGDDASKFLAPQYAALADEAGCGFFDAGSVAQTTPLDGVHLDAENTREIGKA LTPIVRVMLELMoraxella bovis Mbo (AAK53448) (DNA) (SEQ ID NO: 98)ATGAAAAAATCCGCCTTTGCCAAATACTCAGCACTTGCCCTAATGGTTGGGATGTGCCTGCACACCGCTTACGCCAAGGAGTTTAGCCAAGTCATCATTTTTGGGGACAGCTTGTCCGATACAGGTCGCCTAAAAGATATGGTCGCCCGAAAAGATGGCACCCTTGGCAACACCTTACAGCCATCTTTTACCACCAACCCCGACCCTGTATGGTCAAGCTTATTTGCCCAAAGTTATGGCAAAACCGCCAGTCCCAACACGCCTGACAATCCCACTGGCACTAACTATGCCGTGGGCGGAGCTCGCTCTGGCTCGGAGGTCAATTGGAATGGTTTTGTGAATGTACCCTCCACCAAAACGCAAATCACCGACCATTTGACCGCCACAGGTGGCAAAGCCGACCCTAATACCCTGTATGCCATTTGGATTGGCTCTAATGACTTAATTTCAGCTTCTCAAGCCACCACAACAGCCGAAGCCCAAAACGCCATTAAAGGTGCGGTAACTCGCACCGTGATAGACATCGAAACACTCAATCAAGCAGGGGCGACAACCATTTTGGTGCCAAATGTGCCTGATTTGAGCCTCACGCCCCGAGCCATCTATGGCGAAAGCCTCATGGCAGGCGTGCAAGACAAAGCCAAACTCGCCTCAAGTCTGTATAATAGCGGTCTGTTTGAAGCATTAAATCAATCCACCGCCAACATCATCCCTGCCAACACCTTTGCCCTACTCCAAGAAGCGACCACAAATAAAGAAGCCTTTGGTTTTAAAAACACGCAAGGCGTGGCGTGTCAAATGCCCGCTCGTACCACAGGGGCGGATGATGTGGCTTCTACTTCCTTGGCATGTACCAAAGCCAATCTTATAGAAAACGGGGCAAATGACACCTACGCCTTTGCCGATGACATTCACCCATCGGGACGCACGCACCGCATTTTGGCACAGTATTACCGTTCTATCATGGACGCCCCTACTCACATGGGTAAACTCTCAGGCGAGCTTGTCAAAACAGGTTCAGCCCACGACCGTCATGTTTACCGTCAGCTTGACAGGCTTAGTGGCTCACAGCACAGCATTTGGGCAAACGTCTATGCCAGCGACCGTACCGACCCCACCACCCAAATCGGCTTGGACGTGGCAGGTTCATCAAGCCATACAGGGGCGTATCTGAGCCACCAAAACCAAGATTATGTGCTGGATGACACCCTATCATCAGATGTCAAAACCATTGGCATGGGGCTGTATCATCGCCATGACATCGGCAATGTCCGTCTAAAAGGCGTGGCAGGTATCGACCGACTTAGCGTGGATACGCACCGCCATATCGACTGGGAGGGGACAAGCCGTTCGCACACCGCAGATACCACCGCCAGACGTTTTCATGCAGGGCTACAAGCCAGCTATGGCATAGACATGGGCAAAGCCACCGTGCGTCCGCTTATCGGCGTACATGCCCAAAAAGTCAAAGTAAATGACATGACCGAGAGCGAATCAACTTTATCCACCGCCATGCGTTTTGGCGAGCAAGAACAAAAGTCCCTACAAGGCGAGATTGGCGTCGATGTGGCTTATCCGATTAGCCCTGCTTTGACTCTGACGGGCGGTATCGCTCACGCTCATGAGTTTAACGATGATGAACGCACCATTAATGCCACTTTAACCTCCATTCGTGAATACACGAAGGGCTTTAATACAAGCGTTAGCACCGACAAATCTCACGCCACCACCGCTCATCTGGGCGTACAAGGGCAACTTGGCAAGGCAAATATTCATGCAGGCGTTCACGCCACCCACCAAGACAGCGATACAGACGTGGGTGGTTCGCTTGGGGTTCGCTTGATGTTTTA AMoraxella bovis Mbo (AAK53448) (Amino Acid) (SEQ ID NO: 99)MKKSAFAKYSALALMVGMCLHTAYAKEFSQVIIFGDSLSDTGRLKDMVARKDGTLGNTLQPSFTTNPDPVWSSLFAQSYGKTASPNTPDNPTGTNYAVGGARSGSEVNWNGFVNVPSTKTQITDHLTATGGKADPNTLYAIWIGSNDLISASQATTTAEAQNAIKGAVTRTVIDIETLNQAGATTILVPNVPDLSLTPRAIYGESLMAGVQDKAKLASSLYNSGLFEALNQSTANIIPANTFALLQEATTNKEAFGFKNTQGVACQMPARTTGADDVASTSLACTKANLIENGANDTYAFADDIHPSGRTHRILAQYYRSIMDAPTHMGKLSGELVKTGSAHDRHVYRQLDRLSGSQHSIWANVYASDRTDPTTQIGLDVAGSSSHTGAYLSHQNQDYVLDDTLSSDVKTIGMGLYHRHDIGNVRLKGVAGIDRLSVDTHRHIDWEGTSRSHTADTTARRFHAGLQASYGIDMGKATVRPLIGVHAQKVKVNDMTESESTLSTAMRFGEQEQKSLQGEIGVDVAYPISPALTLTGGIAHAHEFNDDERTINATLTSIREYTKGFNTSVSTDKSHATTAHLGVQGQLGKANIHAGVHATHQ DSDTDVGGSLGVRLMFChromobacterium violaceum Cvi (Q7NRP5) (DNA) (SEQ ID NO: 100)ATGCGCTCTATCGTCTGCAAAATGCTGTTCCCTTTGTTGCTGCTGTGGCAGCTGCCCGCCCTGGCCGCCACCGTGCTGGTGTTCGGCGACAGCCTGTCCGCCGGCTACGGCCTGGCCCCGGGCCAGGGATGGGCGGCGCTGCTGGCGCGCGACCTCTCGCCCCGGCACAAGGTGGTCAACGCCAGCGTGTCCGGCGAAACCAGCGCCGGCGGCCTGTCCAGGCTGCCCGACGCGCTCGCCCGCCACCAGCCCGACGTGCTGGTGCTGGAACTCGGCGCCAACGATGGCCTGCGCGGCCTGCCGATGGCTGACATGAGGCGCAACCTGCAGCGGATGATAGACCTGGCCCAGGCGCGCAAGGCCAAGGTGCTGCTGGTGGGCATGGCGCTGCCACCCAACTATGGCCCCCGCTACGGCGCCGAGTTCCGCGCCGTTTATGACGATTTGGCCCGCCGCAACCGCCTGGCCTACGTGCCGCTGCTGGTCGAGGGCTTCGCCGGCGACCTCGGCGCCTTCCAGCCCGACGGCCTGCATCCCCGCGCGGAGAAGCAGGCCACCATGATGCGCACGGTCAAGGCAAAACTGCCAGTGAAATAAChromobacterium violaceum Cvi (Q7NRP5) (Amino Acid) (SEQ ID NO: 101)MRSIVCKMLFPLLLLWQLPALAATVLVFGDSLSAGYGLAPGQGWAALLARDLSPRHKVVNASVSGETSAGGLSRLPDALARHQPDVLVLELGANDGLRGLPMADMRRNLQRMIDLAQARKAKVLLVGMALPPNYGPRYGAEFRAVYDDLARRNRLAYVPLLVEGFAGDLGAFQPDGLHPRAEKQATMMRTVKAKLPVKVibrio vulnificus Vvu (AA007232) (DNA) (SEQ ID NO: 102)ATGTTTTTCCTTTCTAGCGTCGCACACGCAACCGAGAAAGTGTTAATTCTTGGCGACAGCCTAAGTGCAGGATACAACATGTCTGCAGAGCAGGCTTGGCCTAATTTGTTACCAGAAGCATTGAATACATACGGAAAAAACGTAGAAGTGATCAACGCCAGTATCTCTGGAGACACAACCGGCAATGGACTATCTCGTCTGCCTGAGTTGTTAAAAACGCACTCACCAGACTGGGTGCTTATTGAGTTGGGTGCCAATGATGGCTTGCGAGGTTTCCCGCATAAAGTGATCTCTTCAAACCTTTCGCGAATGATTCAACTCAGTAAAGCCTCAGACGCTAAAGTCGCATTGATGCAAATTCGTGTACCGCCTAACTATGGCAAGCGCTACACCGATGCATTTGTCGAACTCTACCCTACGCTTGCTGAACATCACCAAGTCCCGTTGCTCCCCTTTTTCTTAGAGGAAGTGATCGTGAAACCGGAATGGATGATGCCTGATGGCTTACACCCAATGCCCGAAGCTCAGCCTTGGATCGCTCAATTTGTTGCAAAAACGTTTTACAAACATCTCTAA Vibrio vulnificus Vvu (AA007232) (Amino Acid) (SEQ ID NO: 103)MFFLSSVAHATEKVLILGDSLSAGYNMSAEQAWPNLLPEALNTYGKNVEVINASISGDTTGNGLSRLPELLKTHSPDWVLIELGANDGLRGFPHKVISSNLSRMIQLSKASDAKVALMQIRVPPNYGKRYTDAFVELYPTLAEHHQVPLLPFFLEEVIVKPEWMMPDGLHPMPEAQPWIAQFVAKTFYKHLRalstonia eutropha Reu (ZP00166901) (DNA) (SEQ ID NO: 104)ATGCCATTGACCGCGCCGTCTGAAGTCGATCCGCTGCAAATCCTGGTCTATGCCGATTCGCTTTCGTGGGGCATCGTGCCCGGCACCCGCCGGCGGCTTCCCTTCCCGGTTCGCTGGCCAGGCCGGCTCGAACTCGGCCTGAACGCCGACGGCGGCGCCCCGGTCCGCATCATCGAGGACTGCCTGAACGGCCGGCGCACCGTCTGGGACGACCCATTCAAACCGGGCCGCAACGGCTTGCAAGGGCTGGCGCAGCGCATCGAGATCCATTCCCCGGTGGCGCTCGTGGTTTTGATGCTGGGCAACAACGATTTCCAGTCCATGCATCCGCACAACGCCTGGCATGCGGCACAGGGCGTCGGCGCGCTGGTCCACGCCATCCGGACGGCGCCGATCGAACCGGGAATGCCGGTGCCGCCGATCCTGGTGGTGGTGCCGCCGCCGATCCGCACGCCCTGCGGGCCGCTCGCGCCCAAGTTCGCCGGCGGCGAACACAAGTGGGCAGGCCTGCCCGAGGCGCTGCGCGAACTGTGCGCCACTGTCGACTGCTCGCTGTTCGATGCGGGTACCGTGATCCAGAGCAGTGCCGTCGACGGCGTACACCTTGACGCCGATGCCCATGTCGCCCTGGGCGATGCCCTGCAACCGGTCGTTCGTGCGCTGCTCGCCGAATCCTCGGGACATCCCTCCTAARalstonia eutropha Reu (ZP00166901)  (Amino Acid) (SEQ ID NO: 105)MPLTAPSEVDPLQILVYADSLSWGIVPGTRRRLPFPVRWPGRLELGLNADGGAPVRIIEDCLNGRRTVWDDPFKPGRNGLQGLAQRIEIHSPVALVVLMLGNNDFQSMHPHNAWHAAQGVGALVHAIRTAPIEPGMPVPPILVVVPPPIRTPCGPLAPKFAGGEHKWAGLPEALRELCATVDCSLFDAGTVIQSSAVDGVHLDADAHVALGDALQPVVRALLAESSGHPSSalmonella typhimurium Stm (AAC38796) (DNA) (SEQ ID NO: 106)ATGACCCAAAAGCGTACCCTGCTAAAATACGGCATACTCTCGCTGGCGCTGGCCGCGCCATTATCTGCCTGTGCGTTTGACTCTCTTACGGTGATTGGCGATAGCCTTAGCGATACCGGTAATAACGGTCGCTGGACCTGGGATAGTGGTCAAAATAAGCTCTACGACGAACAGTTGGCCGAACGATATGGGCTGGAATTAAGCCCTTCCAGCAATGGCGGCTCTAATTATGCCGCCGGCGGCGCGACGGCGACCCCGGAATTAAACCCGCAGGATAATACCGCGGATCAGGTACGGCAGTGGCTTGCCAAAACGGGGGGAAAAGCCGACCACAACGGTTTGTATATTCACTGGGTCGGCGGAAACGATCTGGCGGCGGCCATCGCGCAACCAACCATGGCACAGCAAATAGCCGGTAATAGCGCCACTAGCGCGGCGGCGCAGGTAGGGCTGTTACTGGATGCCGGCGCCGGGCTGGTCGTGGTGCCAAACGTACCGGATATTAGTGCGACGCCAATGCTTCTGGAGGCGGTAATCACCGCTGGGCTGGGCGCAGCGGCGCCCCCGGCGCTAAAAGCGGCGTTAGATGCGCTGGCGGAGGGCGCTACGCCCGATTTCGCCAGTCGGCAACAGGCGATCCGCAAGGCGCTGCTGGCGGCGGCTGCAACGGTAAGCAGCAATCCATTTATTCAGCAACTGCTCGTTGAACAACTGCTGGCGGGCTATGAAGCGGCGGCAGGGCAGGCGTCAGCTCTGACCGATTATTATAATCAGATGGAAGAGAAGGGGCTGGAGCAACACGGCGGCAATATAGCCCGTGCCGATATCAACGGCCTCTTTAAGGAAATTCTTGCCAACCCGCAGGCGTTTGGTCTGACAAATACCGTAGGTATGGCCTGCCCGCCTGGCGTATCCGCTTCGGCGTGCTCCTCGGCAATGCCTGGATTTAATGCGTCGCAGGACTATGTGTTTGCCGATCATTTACATCCCGGTCCGCAGGTCCATACCATTATTGCGCAATATATTCAGTCGATCATTGCCGCGCCGGTACAGGCGACATACCTGAACCAAAGCGTTCAGTCGATGGCGCAAGGCAGTCGTACCACGCTTGACAGCCGTTATCAGCAGCTTCGCCAGGGGGAAAATCCTGTTGGTTCGCTGGGCATGTTCGGCGGATACAGCGGGGGATATCAACGTTATGATAATAATGAGGCCGACGGGAACGGTAATCATAATAATCTGACGGTTGGCGTCGATTATCAGCTTAACGAGCAGGTTCTGCTGGGAGGGCTGATAGCCGGTTCTCTGGATAAGCAACATCCTGACGATAATTATCGTTATGATGCCCGCGGTTTTCAGGCCGCCGTATTCAGCCATTTACGCGCCGGTCAGGCGTGGCTGGATAGCGATTTACACTTTCTGTCCGCTAAATTCAGTAACATTCAGCGCAGTATAACGCTCGGTGCGCTAAGACGGGTGGAAGAGGGCGAAACCAACGGTCGGCTGTCGGGCGCGAGCTTAACCAGCGGTTATGATTTTGTCATGGTGCCGTGGTTAACGACCGGACCGATGCTGCAATATGCATGGGATTACAGCCACGTTAATGGTTATAGCGAGAAGCTCAATACCAGTACATCAATGCGTTTTGGTGACCAAAACGCCCATTCGCAGGTGGGTAGCGCGGGTTGGCGTCTGGATCTTCGCCACAGCATCATTCACTCCTGGGCGCAGATTAATTATCGCCGTCAGTTTGGCGATGATACGTATGTGGCGAACGGCGGCCTTAAATCGACCGCGCTGACGTTTAGCCGCGACGGAAAAACGCAGGATAAAAACTGGGTTGATATCGCGATTGGCGCAGATTTTCCGCTGTCGGCAACGGTGTCCGCTTTCGCCGGGCTGTCGCAAACGGCAGGGTTAAGCGATGGCAATCAAACCCGTTATAACGTT GGGTTTAGCGCCCGATTTTAASalmonella typhimurium Stm (AAC38796) (Amino Acid) (SEQ ID NO: 107)MTQKRTLLKYGILSLALAAPLSACAFDSLTVIGDSLSDTGNNGRWTWDSGQNKLYDEQLAERYGLELSPSSNGGSNYAAGGATATPELNPQDNTADQVRQWLAKTGGKADHNGLYIHWVGGNDLAAAIAQPTMAQQIAGNSATSAAAQVGLLLDAGAGLVVVPNVPDISATPMLLEAVITAGLGAAAPPALKAALDALAEGATPDFASRQQAIRKALLAAAATVSSNPFIQQLLVEQLLAGYEAAAGQASALTDYYNQMEEKGLEQHGGNIARADINGLFKEILANPQAFGLTNTVGMACPPGVSASACSSAMPGFNASQDYVFADHLHPGPQVHTIIAQYIQSIIAAPVQATYLNQSVQSMAQGSRTTLDSRYQQLRQGENPVGSLGMFGGYSGGYQRYDNNEADGNGNHNNLTVGVDYQLNEQVLLGGLIAGSLDKQHPDDNYRYDARGFQAAVFSHLRAGQAWLDSDLHFLSAKFSNIQRSITLGALRRVEEGETNGRLSGASLTSGYDFVMVPWLTTGPMLQYAWDYSHVNGYSEKLNTSTSMRFGDQNAHSQVGSAGWRLDLRHSIIHSWAQINYRRQFGDDTYVANGGLKSTALTFSRDGKTQDKNWVDIAIGADFPLSATVSAFAGLSQTAGLSDGNQTRYNV GFSARF

In total, nine of the new “GDSL”-type esterases were identified in 6metagenomic libraries and BRAIN's esterase/lipase library. Eight ofthese genes were heterologously expressed in E. coli and the resultingenzymes analyzed for activity in the assays described herein. Thecharacterization of these enzymes for perhydrolase activity revealedthat one displayed the desired activity. A second one was predicted toshow this activity due to the presence of amino acids conserved amongthis group of enzymes.

Comparison of the sequences of enzymes for which the presence or absenceof the desired perhydrolase activity was determined led to theidentification of 19 amino acid positions which were conserved among theenzymes which displayed the desired perhydrolase activity. Thus, it iscontemplated that these conserved amino acids are essential for theperhydrolase reaction and/or is a structural feature of perhydrolaseenzymes.

One of the identified structural motifs (“G/ARTT”) conserved amongesterases with the desired perhydrolase activity was used to designdegenerate primers which provided the means to focus the screening ontrue perhydrolases among “GDSL”-type esterases. Indeed, the use of these“G/ARTT” primers led to the identification of enzymes with the desiredperhydrolase activity from the metagenome. However, it is not intendedthat the use of the metagenome be limited to any particular assaymethod. Indeed, it is contemplated that the metagenome be searched byassaying for a particular enzyme activity or activities desired (e.g.,perhydrolysis and/or acyltransferase (cofactor dependent or independent)activity). In addition, screening using poly and/or monoclonal anti-seradirected against a protein of interest finds use in the presentinvention. In additional embodiments, the metagenome is searched usingdegenerate primer sets based on the sequence of the protein of interest.

In addition, the knowledge of the structure/function relationship ofperhydrolases allowed searching for these enzymes in genome sequences ofcultivable microorganisms. Of 16 “GDSL”-type esterases identified indifferent bacterial isolates, the corresponding genes of 10 enzymes wereamplified and heterologously expressed in E. coli. The resulting enzymesamples of seven clones were analyzed using the assays described herein.Of five samples characterized to date, 4 enzymes indeed showed thedesired activity and all results confirmed the proposed relationshipbetween primary structural determinants and the function ofperhydrolases. Thus, an enzyme library of 19, “GDSL”-type esterasescomprising at least 6 perhydrolases with the desired perhydrolaseactivity was set up. The identified correlation between the structureand function of perhydrolases provides a definition of the sequencespace used by enzymes with the desired perhydrolase activity.

Comparisons were made of protein sequences of enzymes for which theabsence or presence of the desired perhydrolase activity. This revealeda correlation between the presence of certain amino acids and thecapability to perform perhydrolase reactions. This knowledge was used toidentify enzymes containing these conserved amino acids in sequencedgenomes from cultivable microorganisms. The following enzymes wereidentified and experiments to amplify the genes from the genomic DNA ofthe corresponding strains using specific primers were performed.

TABLE 1 “GDSL”-type Esterases with a “GRTT”-Motif From BacterialIsolates Protein Ac- Expression Isolate Identifier ronym Amplicon VectorSinorhizobium Sma1993 Sme I yes pLO_SmeI meliloti Sinorhizobium Q92XZ1Sme II yes pET26_SmeII meliloti Sinorhizobium Q9EV56 Sme III yespET26_SmeIII meliloti Agrobacterium Q9KWB1 Arh I no — rhizogenesAgrobacterium Q9KWA6 Arh II no — rhizogenes Agrobacterium AAD02335 AtuIII yes pET26_AtuIII tumefaciens Mesorhizobium Q98MY5 Mlo I yespET26_Mlo loti Mesorhizobium ZP_00197751 Mlo II no — loti RalstoniaQ8XQI0 Rso no — solanacearum Ralstonia ZP_00166901 Reu yes n.d. eutrophaMoraxella bovis AAK53448 Mbo yes pET26_Mbo Burkholderia ZP_00216984 Bceno — cepacia Chromobacterium Q7NRP5 Cvi yes pET26_Cvi violaceumPirellula sp. NP_865746 Psp n.d. n.d. Vibrio vulnificus AA007232 Vvu yespET26_Vvu Salmonella AAC38796 Sty yes pET26_Sty typhimurium

In the cases of A. rhizogenes, M. loti (enzyme II), R. solanacearum andB. cepacia no amplicon could be generated. It was thought that this wasprobably due to genetic differences between the strains used in thisinvestigation and those used for the sequencing of the genes depositedin the public domain databases. One reason might be that thecorresponding genes are located on plasmids which are not present in thestrains used in this investigation. However, it is not intended that thepresent invention be limited to any particular mechanism or theory.

The amplicons from all other strains were sequenced. In many cases therewere differences between the sequence from the databases and thesequence determined during the development of the present invention. Bysequencing two clones from independent amplifications, mutationsintroduced by the polymerase could be nearly excluded. The sequences ofthe genes and the deduced amino acid sequences of “GDSL”-type esteraseswith a “GRTT”-motif or variations from bacterial isolates are providedbelow:

SMa1993_(—) Sinorhizobium meliloti (Sme I) (SEQ ID NOS:88 and 89)

Q92XZ1_(—) Sinorhizobium meliloti (Sme II) (SEQ ID NOS:90 and 91)

Q9EV56_(—) Sinorhizobium meliloti (Sme III) (SEQ ID NOS:92 and 93)

AAD02335_(—) Agrobacterium tumefaciens (Atu III) (SEQ ID NOS: 94 and 95)

Q98MY5_(—) Mesorhizobium loti (Mlo I) (SEQ ID NOS:96 and 97)

ZP_(—)00166901_(—) Ralstonia eutropha (Reu) (SEQ ID NOS:104 and 105)

AAK53448_(—) Moraxella bovis (Mbo) (SEQ ID NOS: 98 and 99)

Q7NRP5_(—) Chromobacterium violaceum (Cvi) (SEQ ID NOS:100 and 101)

AA007232_(—) Vibrio vulnificus (Vvu) (SEQ ID NOS:102 and 103)

AAC38796_(—) Salmonella typhimurium (Stm) (SEQ ID NOS:106 and 107)

Q9KWB1_Agrobacterium rhizogenes (Arh I) (SEQ ID NO: 108)MICHKGGEEMRSVLCYGDSNTHGQIPGGSPLDRYGPNERWPGVLRRELGSQWYVIEEGLSGRTTVRDDPIEGTMKNGRTYLRPCLMSHAILDLVIIMLGTNDLKARFGQPPSEVAMGIGCLVYDIRELAPGPGGKPPEIMVVAPPPMLDDIKEWEPIFSGAQEKSRRLALEFEIIADSLEVHFFDAATVASCDPCDGFHINREAHEALGTALAREVEAIGWR (SEQ ID NO: 109)ATGATTTGCCATAAAGGTGGGGAGGAAATGCGGTCAGTCTTATGCTACGGCGACTCGAATACGCACGGCCAGATTCCGGGGGGCTCACCGCTCGACCGATACGGGCCGAACGAGCGCTGGCCTGGCGTTTTGAGACGGGAGCTTGGAAGCCAGTGGTATGTGATCGAGGAGGGCCTGAGTGGCCGCACGACGGTTCGCGACGATCCGATCGAGGGCACGATGAAAAACGGCCGGACCTACCTGCGTCCGTGCCTCATGAGCCACGCGATCCTCGATCTCGTGATTATCATGCTCGGGACGAACGACCTGAAAGCGCGCTTCGGTCAACCGCCATCGGAAGTGGCGATGGGGATCGGCTGCCTCGTCTACGATATCAGGGAGCTGGCGCCCGGACCGGGCGGCAAGCCCCCCGAAATCATGGTGGTTGCTCCGCCGCCGATGCTGGACGATATCAAGGAATGGGAACCCATATTTTCCGGCGCCCAGGAGAAATCCCGGCGTCTCGCGCTTGAGTTTGAAATTATTGCTGATTCGCTTGAAGTACACTTCTTTGACGCCGCGACCGTCGCATCGTGTGATCCTTGCGATGGTTTTCACATCAACCGGGAAGCGCATGAAGCCTTGGGAACAGCGCTTGCCAGGGAAGTGGAGGCGATCGGTTGGAGATGATGA Q9KWA6_Agrobacterium rhizogenes (Arh II)(SEQ ID NO: 110) MAESRSILCFGDSLTWGWIPVPESSPTLRYPFEQRWTGAMAAALGDGYSIIEEGLSARTTSVEDPNDPRLNGSAYLPMALASHLPLDLVIILLGTNDTKSYFRRTPYEIANGMGKLAGQVLTSAGGIGTPYPAPKLLIVSPPPLAPMPDPWFEGMFGGGYEKSLELAKQYKALANFLKVDFLDAGEFVKTDGCDGIHFSAETNITLGHAIAAKVEAIFSQEAKNAAA (SEQ ID NO: 111)ATGGCAGAGAGCCGCTCAATATTATGTTTTGGGGATTCACTCACATGGGGTTGGATTCCGGTACCGGAGTCGTCGCCGACGCTCAGATATCCCTTTGAGCAGCGCTGGACCGGTGCAATGGCTGCGGCACTCGGTGACGGCTATTCAATCATCGAGGAAGGCCTTTCCGCCCGCACGACCAGCGTCGAGGATCCGAACGATCCCAGGCTGAACGGCAGCGCCTACCTGCCGATGGCGCTCGCCAGCCATCTGCCGCTCGATCTCGTCATCATCCTTCTCGGCACCAACGACACCAAGTCCTATTTCCGCCGCACGCCCTATGAGATCGCCAACGGCATGGGCAAGCTTGCCGGACAGGTTCTGACCTCGGCCGGCGGGATCGGCACGCCCTACCCTGCCCCGAAGCTTCTGATCGTTTCGCCGCCGCCGCTCGCTCCCATGCCTGACCCGTGGTTCGAAGGCATGTTCGGTGGCGGTTACGAAAAGTCGCTCGAACTCGCAAAGCAGTACAAGGCGCTCGCCAACTTCCTGAAGGTCGACTTCCTCGACGCCGGCGAGTTTGTAAAGACCGACGGCTGCGATGGAATCCATTTCTCCGCCGAGACGAACATCACGCTCGGCCATGCGATCGCGGCGAAGGTCGAAGCGATTTTCTCACAAGAGGCGAAGAACGCTGCGGCTTAGZP_00197751_Mesorhizobium loti (Mlo II) (SEQ ID NO: 112)MKTILCYGDSLTWGYDAVGPSRHAYEDRWPSVLQGRLGSSARVIAEGLCGRTTAFDDWVAGADRNGARILPTLLATHSPLDLVIVMLGTNDMKSFVCGRAIGAKQGMERIVQIIRGQPYSFNYKVPSILLVAPPPLCATENSDFAEIFEGGMAESQKLAPLYAALAQQTGCAFFDAGTVARTTPLDGIHLDAENTRAIGA GLEPVVRQALGL(SEQ ID NO: 113) ATGAAGACCATCCTTTGTTACGGTGACTCCCTCACTTGGGGCTATGATGCCGTCGGACCCATGAAGACCATCCTTTGTTACGGTGACTCCCTCACTTGGGGCTATGATGCCGTCGGACCCTCACGGCATGCTTATGAGGATCGATGGCCCTCCGTACTGCAAGGCCGCCTCGGTAGCAGTGCGCGGGTGATCGCCGAGGGGCTTTGCGGCCGCACAACTGCGTTTGACGACTGGGTCGCTGGTGCGGACCGGAACGGTGCGCGCATCCTGCCGACGCTTCTTGCGACCCATTCACCGCTTGACCTCGTTATCGTCATGCTCGGGACGAACGACATGAAATCGTTCGTTTGCGGGCGCGCTATCGGCGCCAAGCAGGGGATGGAGCGGATCGTCCAGATCATCCGCGGGCAGCCTTATTCCTTCAATTATAAGGTACCGTCGATTCTTCTCGTGGCGCCGCCGCCGCTGTGCGCTACCGAAAACAGCGATTTCGCGGAAATTTTTGAAGGTGGCATGGCTGAATCGCAAAAGCTCGCGCCGCTTTATGCCGCGCTGGCCCAGCAAACCGGATGCGCCTTCTTCGATGCAGGCACTGTGGCCCGCACGACACCGCTCGACGGTATTCACCTCGATGCTGAAAACACGCGCGCCATTGGTGCCGGCCTGGAGCCGGTGGTCCGCCAAGCGCTTGGATTGTGAQ8XQI0_Ralstonia solanacearum (Rso) (SEQ ID NO: 114)MQQILLYSDSLSWGIIPGTRRRLPFAARWAGVMEHALQAQGHAVRIVEDCLNGRTTVLDDPARPGRNGLQGLAQRIEAHAPLALVILMLGTNDFQAIFRHTAQDAAQGVAQLVRAIRQAPIEPGMPVPPVLIVVPPAITAPAGAMADKFADAQPKCAGLAQAYRATAQTLGCHVFDANSVTPASRVDGIHLDADQHAQLG RAMAQVVGTLLAQ(SEQ ID NO: 115) ATGCAACAGATCCTGCTCTATTCCGACTCGCTCTCCTGGGGCATCATCCCCGGCACCCGCCGGCGCCTGCCGTTCGCCGCCCGCTGGGCCGGGGTCATGGAACACGCGCTGCAGGCGCAAGGGCACGCCGTGCGCATCGTCGAAGACTGCCTCAATGGACGCACCACGGTGCTCGACGATCCCGCGCGGCCGGGGCGCAACGGACTGCAGGGGCTCGCGCAGCGGATCGAAGCGCACGCCCCGCTTGCCCTGGTCATCCTGATGCTCGGCACCAACGACTTCCAGGCGATCTTCCGGCACACCGCCCAGGACGCGGCGCAAGGCGTGGCGCAGCTGGTGCGGGCCATCCGCCAGGCGCCGATCGAACCCGGCATGCCGGTGCCGCCCGTGCTGATCGTGGTGCCGCCGGCCATCACCGCGCCGGCCGGGGCGATGGCCGACAAGTTTGCCGACGCGCAGCCCAAGTGCGCCGGCCTTGCGCAGGCCTATCGGGCAACGGCGCAAACGCTAGGCTGCCACGTCTTCGATGCGAACAGCGTCACGCCGGCCAGCCGCGTGGACGGCATCCACCTCGATGCCGACCAGCATGCGCAGCTGGGCCGGGCGATGGCGCAGGTCGTCGGGACGCTGCTTGCGCAATAAZP_00216984 Burkholderia cepacia (Bce) (SEQ ID NO: 116)ATGACGATGACGCAGAAAACCGTGCTCTGCTACGGCGATTCGAACACGCATGGCACACGCCCGATGACGCATGCTGGCGGACTGGGGCGGTTTGCACGCGAAGAACGCTGGACCGGCGTGCTGGCGCAAACGCTCGGTGCGAGCTGGCGGGTCATTGAAGAAGGGTTGCCCGCGCGTACGACCGTGCATGACGATCCGATCGAAGGCCGGCACAAGAATGGTTTGTCGTATCTGCGCGCGTGCGTCGAAAGCCACTTGCCCGTCGATGTCGTCGTGCTGATGCTCGGGACCAACGATCTGAAGACACGCTTCTCGGTCACGCCCGCCGACATCGCGACATCGGTCGGCGTATTGCTTGCCAAGATCGCTGCGTGCGGCGCCGGTCCGTCCGGTGCGTCACCGAAGCTCGTGCTGATGGCGCCTGCGCCGATCGTCGAGGTCGGATTCCTCGGCGAGATCTTTGCGGGCGGCGCAGCGAAGTCGCGGCAGCTCGCGAAGCGGTACGAACAGGTGGCAAGCGATGCCGGTGCGCACTTTCTCGATGCCGGCGCGATCGTCGAGGTGAGCCCGGTGGATGGCGTTCACTTCGCGGCCGATCAGCATCGTGTGCTCGGGCAGCGGGTCGCTGCCCTTCTGCAGCAGATTGCGTA A (SEQ ID NO: 117)MTMTQKTVLCYGDSNTHGTRPMTHAGGLGRFAREERWTGVLAQTLGASWRVIEEGLPARTTVHDDPIEGRHKNGLSYLRACVESHLPVDVVVLMLGTNDLKTRFSVTPADIATSVGVLLAKIAACGAGPSGASPKLVLMAPAPIVEVGFLGEIFAGGAAKSRQLAKRYEQVASDAGAHFLDAGAIVEVSPVDGVHFAADQ HRVLGQRVAALLQQIANP_865746 Pirellula sp (Psp) (SEQ ID NO: 118)MHSILIYGDSLSWGIIPGTRRRFAFHQRWPGVMEIELRQTGIDARVIEDCLNGRRTVLEDPIKPGRNGLDGLQQRIEINSPLSLVVLFLGTNDFQSVHEFHAEQSAQGLALLVDAIRRSPFEPGMPTPKILLVAPPTVHHPKLDMAAKFQNAETKSTGLADAIRKVSTEHSCEFFDAATVTTTSVVDGVHLDQEQHQALG TALASTIAEILADC(SEQ ID NO: 119) ATGCATTCAATCCTCATCTATGGCGATTCTCTCAGTTGGGGAATCATTCCCGGCACGCGTCGTCGCTTCGCGTTCCATCAGCGTTGGCCGGGCGTCATGGAGATTGAACTGCGACAAACTGGAATCGATGCCCGCGTCATCGAAGACTGCCTCAATGGCCGACGAACCGTCTTGGAAGATCCAATCAAACCCGGACGCAATGGCCTGGATGGTTTGCAGCAACGGATCGAAATCAATTCACCTCTGTCACTGGTCGTGCTCTTTCTGGGGACCAACGATTTCCAGTCCGTCCACGAATTCCATGCCGAGCAATCGGCACAAGGACTCGCACTGCTTGTCGACGCCATTCGTCGCTCCCCTTTCGAACCAGGAATGCCGACACCGAAAATCCTGCTTGTCGCACCACCGACGGTTCACCACCCGAAACTTGATATGGCGGCGAAGTTCCAAAACGCGGAAACGAAATCGACGGGACTCGCAGATGCGATTCGCAAGGTCTCAACAGAACACTCCTGCGAATTCTTCGATGCGGCCACGGTCACCACAACAAGTGTCGTCGACGGAGTCCATCTCGATCAAGAACAACATCAAGCACTCGGTACCGCACTGGCATCGACAATCGCTGAAATACTAGCAGACTGTTGA

As indicated above, the above sequences are the protein sequences andthe coding sequences of “GDSL-type” esterases with a “GRTT”-motif orsimilar motifs from different bacterial isolates. The DNA sequencesrepresent the target-DNA from which specific primers were deduced. Allamplicons were ligated as NdeI/XhoI-fragments to pET26 therebyeliminating the pelB-leader sequence of this vector. All of the“GDSL-type” esterases from these isolates were expressed in E. coliRosetta (DE3) at 28° C. The expression was induced by addition of 100 μMIPTG at an O.D.₅₈₀=1 and the cells were harvested 20 h after induction.Only the cells expressing the enzymes from M. bovis and S. typhimuriumwere collected 4 h after induction, since previous experiments had shownthat the highest activity could be obtained at this point of time. Table2 summarizes the expression experiments.

TABLE 2 Expression and Characterization of “GDSL”-type Esterases FromBacterial Isolates for Perhydrolase Activity Per- Expres- Solu- hydro-sion bil- Activ- lase GRTT- Strain Enzyme Level² ity³ ity ⁴ ActivityMotif S. meliloti Sme I +++ ++ 5770.0 yes ARTT S. meliloti Sme II ++++++ 85.0 yes GRTT S. meliloti Sme III +++ ++ 746.5 n.d. GRTT A.tumefaciens Atu III n.d⁵. n.d. n.d. n.d. GRTT M. loti Mlo I +++ ++1187.3 yes GRTT M. bovis ¹ Mbo + n.d. 25.2 yes ARTT C. violaceum Cvi + +2422.7 n.d. GETS V vulnificus Vvu n.d. n.d. n.d. n.d. GDTT R. eutrophaReu n.d. n.d. n.d. n.d. GRRT S. typhimurium ¹ Sty + n.d. 17.2 no SRTT¹outer membrane localized autotransporter protein ²expression level: +moderate overexpression; ++ strong overexpression; +++ very strongoverexpression as judged from SDS-PAGE-analysis ³as judged bySDS-PAGE-analysis ⁴ towards p-nitrophenyl butyrate ⁶not determined

With the exception of the enzyme from S. typhimurium, all other enzymestested showed the desired perhydrolase activity, confirming thecorrelation between the presence of certain conserved amino acids an thecapability to perform perhydrolase reactions. Although the enzyme fromS. typhimurium contains the GRTT-motif, it is different from the otherenzymes by the location of this motif downstream from block V. In allother enzymes, this motif is located between block I and III, indicatingthat it might have a different function in the enzyme from S.typhimurium. Thus, the absence of perhydrolase activity in the enzymefrom S. typhimurium also supports the identifiedstructure/function-relationship of the perhydrolases provided by thepresent invention.

Screening of New “GDSL-Type” Esterases in Metagenome Libraries

i) Library S279

-   -   The full-length sequence of the gene from clone M75bA2 was        completed, as provided below.

1 tgggcggttt cgcggagtcg agcagggaga gatgctcctg ggtcgtacga gttggtacgg   g  r  f   r  g  v   e  q  g   e  m  l  l   g  r  t   s  w  y 61aggcatcgtt gaagatctca cgcctgcttg aatgcgcgcg gatatggaac ggaccggccgg  g  i  v   e  d  l   t  p  a   —  m  r  a   d  m  e   r  t  g 121cgctggcgat cggtgtcggc gtggggctgg cgagcctgag cccggtcgcg ctggcgacgcr  a  g  d   r  c  r   r  g  a   g  e  p  e   p  g  r   a  g  d 181cgccgcgggg caccgtgccg gtgttcaccc gatcggggac agcctgacgg acgagtattta  a  a  g   h  r  r   g  v  h   p  i   g  d   s  l   t   d  e  y 241tgagccgttc ttccagtggg ggttctgcgg gaagtcgtgg gccgagattt tggtggagacf  e  p  f   f  q  w   g  f  c   g  k  s  w   a  e  i   l  v  e 301ggggcgggcg agcatgggcc cgacggcgca gcaggcgggg atcagcgagc cggagggatgt  g  r  a   s  m  g   p  t  a   q  q  a  g   i  s  e   p  e  g 361gtcggatccg cggaacacgg ggtatcagca caactgggcg cggtactcgt ggagctcctcw  s  d  p   r  n  t   g  y  q   h  n  w  a   r  y  s   w  s  s 421agacgcgctg accgaggagt cgccgggggc gacgctgagc gtgctgcttg gggcggagtas  d  a  l   t  e  e   s  p  g   a  t  l  s   v  l  l   g  a  e 481cgcggtggtg ttcattggga ccaacgactt caatccgtcg tggccggcgt atcagagcgty  a  v  v   f  i   g   t  n  d    f  n  p  s   w  p  a   y  q  s 541gtatctgagc cagtggagcg acgagcagat cgacacgtac gtgaacgggg tggtgcagaav  y  l  s   q  w  s   d  e  q   i  d  t  y   v  n  g   v  v  q 601catcgcgcag atggtggact cgctgaagtc ggtcggggcg aaggtggtgc ttgcgccgccn  i  a  q   m  v  d   s  l  k   s  v  g  a   k  v  v   l  a  p 661ggtggatttt cagttcgcgg ggttcctgcg gaactcatgc ccggatccga tgctgcgcgap  v  d  f   q  f  a   g  f  l   r  n  s  c   p  d  p   m  l  r 721gcaggcgggt attctgacac ggaagtgcca cgaccgggtg cggtcgatgg cgcggcagaae  q  a  g   i  l  t   r  k  c   h  d  r  v   r  s  m   a  r  q 781gcacgtggtg ttcgtggaca tgtggcggct gaaccgcgat ttgttcggca acgggttcgck  h  v  v   f  v  d   m  w  r   l  n  r  d   l  f  g   n  g  f  841gatcagctac ggccttcgga acacggtgcg cgtgggggac tcggagatcg ggctgcaacta  i  s  y   g  l  r   n  t  v   r  v  g  d   s  e  i   g  l  q 901ggccgggctg acgggatcgg cggggctggt tccggacggg atccatccgc agcgggtggtl  a  g  l   t  g  s   a  g  l   v  p   d  g   i  h  p    q  r  v 961gcaggggatc tgggcgaatg cgttcatcgt gggtctgaac gcgcatgggg cgaacatcgcv  q  g  i   w  a  n   a  f  i   v  g  l  n   a  h  g   a  n  i 1021gcccatcggc gaggcggaga tgtgcgcgat ggggggggtc gtgtacgggg gaacggacaca  p  i  g   e  a  e   m  c  a   m  g  g  v   v  y  g   g  t  d 1081gctggcgaac ttcctgccgc cggtcgcggg ctacgtggag gacttccgca acgcgggggat  l  a  n   f  l  p   p  v  a   g  y  v  e   d  f  r   n  a  g 1141cttcgtgtgc acggcggact tcaaccatga ccttggcgtg acgccgacgg acatcttcgcd  f  v  c   t  a  d   f  n  h   d  l  g  v   t  p  t   d  i  f 1201gttcatcaac gcgtggttca tgaatgatcc ctcggcgcgg atgagcaacc cggagcacaca  f  i  n   a  w  f   m  n  d   p  s  a  r   m  s  n   p  e  h 1261gcagatcgag gacatcttcg tgtttctgaa tctgtggctg gtggggtgct gaggcagagtt  q  i  e   d  i  f   v  f  l   n  l  w  i   v  g  c   —  g  r 1321gggaaggggg tcagcccact tcgcgcgtct ggaagaggat gacggcgacg gagaggaagav  g  r  g   s  a  h   f  a  r   l  e  e  d   d  g  d   g  e  e

In the sequence of S279_M75bA2 provided above (DNA, SEQ ID NO:80; andamino acid sequence, SEQ ID NO:81), the coding sequence running fromposition 104 through 1312 is shown on a grey background. Conservedstructural motifs are shown underlined and in bold.

The derived amino acid sequence showed the highest homology to ahypothetical protein (Y17D7A.2) from Caenorhabditis elegans (BlastP2;swisspir), although with a very high E-value of 2.5 (i.e., indicating anon-reliable hit). The fact that no esterase is among the homologousproteins identified by the BlastP2-analysis indicates that this enzymeis a rather unusual “GDSL-type” esterase. Furthermore, the enzyme ischaracterized by unusually long peptides between the N-terminus and the“GDSL”-motif and the “DXXH”-motif of block V (containing the active siteaspartic acid and histidine) and the C-terminus. The very C-terminalsequence shows similarity to a membrane lipoprotein lipid attachmentsite. A corresponding signal sequence of lipoproteins was notidentified. The gene encoding M75bA5 was amplified but no furtherefforts were taken for this enzyme since it did not have the conservedamino acids typical of the perhydrolase of the present invention.

ii) Library S248

The clone carrying the sequence-tag SP7_(—)3j5h which could have beenpart of a gene encoding a “GDSL”-type esterase was identified (M31bA11),and the sequence was elongated. This facilitated the determination thatthis sequence did not encode a “GDSL-type” esterase, because block Vcould not be identified. The generation of this amplicon can beexplained by an “unspecific” hybridization of primer 5h with the firstmismatches at nucleotides 10, 14 and 15 from the 3-terminus of theprimer. The sequence showed the highest homology to a hypotheticalprotein (KO3E5.5) from Caenorhabditis elegans with an E-value of 1.6,indicating a non-reliable hit. The sequence-tag from clone S248_M31bA11is provided below.

  1 cggaattatc atgctgggtt ttaatgacca gcgcgagagg atcaacgaca acctcgatta   r  n  y   h  a  g   f  -  -  p   a  r  e   d  q  r   q  p  r  l    g  i  i   m  l  g   f  n  d   q  r  e  r   i  n  d   n  l  d     e  l   s  c  w  v   l  m  t   s  a  r   g  s  t  t   t  s  i  61ctgggacgcc taccactccg tcctgggcga gagacagttt tattccggca attccaagat  l  g  r   l  p  l   r  p  g  r   e  t  v   l  f  r   q  f  q  dy  w  d  a   y  h  s   v  l  g    e r  q  f   y  s  g   n  s  k t  g  t  p   t  t  p   s  w  a    r d  s   f  l  p  a   i  p  r 121gttcgtcccc atcaccaaga tcgcggtgaa ggcgcgcaag acccggttca ccaatcagat  v  r  p   h  h  q   d  r  g  e   g  a  q   d  p  v   h  q  s  dm  f  v  p   i  t  k   i  a  v   k  a  r  k   t  r  f   t  n  q c  s  s   p  s  p  r   s  r  -   r  r  a   r  p  g  s   p  i  r

181 ttttcctcag tccggccgca acgtcgatgt caccaccacg gacggcacac tcccccacgc  f  s  s   v  r  p   q  r  r  c   h  h  h   g  r  h   t  p  p  ri  f  p  q   s  g  r   n  v  d   v  t  t  t   d  g  t   l  p  h f  f  l   s  p  a  a   t  s  m   s  p  p   r  t  a  h   s  p  t

241 caccatgtcc ctggtcgagc actacatccg ggcctgccgc ctgcgcaccc agatcgttcc  h  h  v   p  g  r   a  l  h  p   g  l  p   p  a  h   p  d  r  sa  t  m  s   l  v  e   h  y  i   r  a  c  r   l  r  t   q  i  v p  p  c   p  w  s  s   t  t  s   g  p  a   a  c  a  p   r  s  f 301ggccctgatc gttaacggcg attgcgaagg catgtacagc atctatgtcg gctggtcgaa  g  p  d   r  -  r   r  l  r  r   h  v  q   h  l  c   r  l  v  ep  a  l  i   v  n  g   d  c  e   g  m  y  s   i  y  v   g  w  s r  p  -   s  l  t  a   i  a  k   a  c  t   a  s  m  s   a  g  r 361aaccaccaag catgttgttt cacgtgaaac aaagccggtc gaaagcgacg gcatggaatt  n  h  q   a  c  c   f  t  -  n   k  a  g   r  k  r   r  h  g  ik  t  t  k   h  v  v   s  r  e   t  k  p  v   e  s  d   g  m  e k  p  p   s  m  i  f   h  v  k   q  s  r   s  k  a  t   a  w  n 421tcccgaactg ggcgaagccg acgacatcac cgaagaaacg cttgagtgtg gccttcccga  s  r  t   g  r  s   r  r  h  h   r  r  n   a  -  v   w  p  s  rf  p  e  l   g  e  a   d  d  i   t  e  e  t   l  e  c   g  l  p f  p  n   w  a  k  p   t  t  s   p  k  k   r  l  s  v   a  f  p 481catcgaattg atctcggacg ccgatcttct cgtccttcca ccagcgccga caacattcca  h  r  i   d  i  g   r  r  s  s   r  p  s   t  s  a   d  n  i  p d  i  e  l   i  s  d   a  d  l   l  v  l  p   p  a  p   t  t  f  t  s  n   -  s  r  t   p  i  f   s  s  f   h  q  r  r   q  h  s  541aggcgcttga gatgggcggg ttcggtcacg atcttgcgcc gtggacaagg gcaaggtccg  r  r  l   r  w  a   g  s  v  t   i  l  r   r  g  q   g  q  g  pq  g  a  -   d  g  r   v  r  s   r  s  c  a   v  d  k   g  k  v k  a  l   e  m  g  g   f  g  h   d  l  a   p  w  t  r   a  r  s 601cagatgatcg acgaggcgcg atcaccgaga tgccgcgacg atctgtcgac gctatgtcac  q  m  i   d  e  a   r  s  p  r   c  r  d   d  l  s   t  l  c  h r  r  -  s   t  r  r   d  h  r   d  a  a  t   i  c  r   r  y  v  a  d  d   r  r  g  a   i  t  e   m  p  r   r  s  v  d   a  m  s  661cagcgcatgt ccgacggtgg aatgcaagac aggtnggntn gatcgggg (SEQ ID NO: 120)  q  r  m   s  d  g   g  m  q  d   r  ?  ?   ?  s  g (SEQ ID NO: 121) t  s  a  c   p  t  v   e  c  k   t  g  ?  ?   d  r   (SEQ ID NO: 122)  p  a  h   v  r  r  w   n  a  r   q  ?  ?   ?  i  g  (SEQ ID NO: 123) 

In the above sequence-tag of the clone S248_M31bA11, the primers 3j and5h are indicated. Hybridization between primer and template is indicatedby arrows, mismatches by open circles. Putative conserved structuralmotifs are indicated in bold and underlined.

Several further sequence-tags were generated using different primerpairs of the primers 2 and 5 but none turned out to encode a “GDSL”-typeesterases. The screening of this library was completed.

iii) Library M091

The elongation of the amplicon SP3_(—)1j5h, which was identified in theinsert-DNA of clone M24dG12 proved that the corresponding sequence doesnot encode a “GDSL”-type esterase. Whereas the sequence encoding aputative block V (DGTHP; SEQ ID NO:124) was found, the correspondingsequence encoding block I was missing. The amplicon was generated due toan “unspecific” hybridization of primer 1j with the first mismatches atpositions 5, 10, 11 and 12 from the 3′-terminus of the primer. Thesequence-tag of clone M091_M24dG12 s shown below:

   1 gcctgatggc ttcgagttcg tcgaattcac ctcgccccag cccggcgtgc tggaggcggt     a  -  w   l  r  v   r  r  i  h   l  a  p   a  r  r   a  g  g  g    p  d  g   f  e  f   v  e  f   t  s  p  q   p  g  v   l  e  a    l  m   a  s  s  s   s  n  s   p  r  p   s  p  a  c   w  r  r   61gtttgaaaag ctgggtttca ccctggtcgc caagcaccgg tccaaggatg tggtgctgta  v  -  k   a  g  f   h  p  g  r   q  a  p   v  q  g   c  g  a  v v  f  e  k   l  g  f   t  l  v   a  k  h  r   s  k  d   v  v  l c  l  k   s  w  v  s   p  w  s   p  s  t   g  p  r  m   w  c  c  121ccgccagaac ggcatcaact tcatcctgaa ccgcgagccc cacagccagg ccgcctactt  p  p  e   r  h  q   l  h  p  e   p  r  a   p  q  p   g  r  l  ly  r  q  n   g  i  n   f  i  l   n  r  e  p   h  s  q   a  a  y t  a  r   t  a  s  t   s  s  -   t  a  s   p  t  a  r   p  p  t  181tggtgccgag catggcccct ccgcctgtgg cctggccttc cgtgtgaagg atgcgcataa  w  c  r   a  w  p   l  r  l  w   p  g  l   p  c  e   g  c  a  -f  g  a  e   h  g  p   s  a  c   g  l  a  f   r  v  k   d  a  h l  v  p   s  m  a  p   p  p  v   a  w  p   s  v  -  r   m  r  i  241ggcttataac cgcgcgctgg aactgggcgc ccagcccatc gagatcccca ccggccccat  g  l  -   p  r  a   g  t  g  r   p  a  h   r  d  p   h  r  p  hk  a  y  n   r  a  l   e  l  g   a  q  p  i   e  i  p   t  g  p r  l  i   t  a  r  w   n  w  a   p  s  p   s  r  s  p   p  a  p 

 301 ggaactgcgc ctgcccgcca tcaagggcat tggcggcgcc gcctctgtat ttgatcgacc  g  t  a   p  a  r   h  q  g  h   w  r  r   r  l  c   i  -  s  tm  e  l  r   l  p  a   i  k  g   i  g  g  a   a  s  v   f  d  r w  n  c   a  c  p  p   s  r  a   l  a  a  p   p  l  y   l  i  d

 361 gctttgaaga cggcaagtcc atctacgaca tcgacttcga gttcatcgaa ggcgtggacc  a  l  k   t  a  s   p  s  t  t   s  t  s   s  s  s   k  a  w  t p  l  -  r   r  q  v   h  l  r   h  r  l  r   v  h  r   r  r  g r  f  e   d  g  k  s   i  y  d   i  d  f   e  f  i  e   g  v  d  421gccgccccgc ggggcatggc ctgaacgaga tcgatcacct cacgcacaac gtgtaccggg  a  a  p   r  g  m   a  -  t  r   s  i  t   s  r  t   t  c  t  g p  p  p  r   g  a  w   p  e  r   d  r  s  p   h  a  q   r  v  p r  r  p   a  g  h  g   l  n  e   i  d  h   l  t  h  n   v  y  r  481gccgcatggg cttctgggcc aacttctacg aaaagctgtt caacttccgc gaaatccgct  a  a  w   a  s  g   p  t  s  t   k  s  c   s  t  s   a  k  s  a g  p  h  g   l  l  g   q  l  l   r  k  a  v   q  l  p   r  n  p g  r  m   g  f  w  a   n  f  y   e  k  l   f  n  f  r   e  i  r  541acttcgacat ccagggcgaa tacacgggcc tgacctccaa ggccatgacc gcgcccgacg  t  s  t   s  r  a   n  t  r  a   -  p  p   r  p  -   p  r  p  t l  l  r  h   p  g  r   i  h  g   p  d  l  q   g  h  d   r  a  r y  f  d   i  q  g  e   y  t  g   l  t  s   k  a  m  t   a  p  d  601gcaagattcg catcccgctg aacgaagagt ccaagcaggg cggcggccag atcgaagaat  a  r  f   a  s  r   -  t  k  s   p  s  r   a  a  a   r  s  k  nr  q  d  s   h  p  a   e  r  r   v  q  a  g   r  r  p   d  r  r g  k  i   r  i  p  l   n  e  e   s  k  q   g  g  g  q   i  e  e  661ttttgatgca attcaacggc gagggcattc agcacatcgc gctgatctgc gacaacctgc  f  -  c   n  s  t   a  r  a  f   s  t  s   r  -  s   a  t  t  ci  f  d  a   i  q  r   r  g  h   s  a  h  r   a  d  l   r  q  p f  l  m   q  f  n  g   e  g  i   q  h  i   a  l  i  c   d  n  l  721tggacgtggt ggacaagctg ggcatggccg gcgtgcagct ggccaccgcg cccaacgagg  w  t  w   w  t  s   w  a  w  p   a  c  s   w  p  p   r  p  t  ra  g  r  g   g  q  a   g  h  g   r  r  a  a   g  h  r   a  q  r l  d  v   v  d  k  l   g  m  a   g  v  q   l  a  t  a   p  n  e  781tctattacga aatgctggac acccgcctgc ccggccacgg ccagccggtg cccgagctgc  s  i  t   k  c  w   t  p  a  c   p  a  t   a  s  r   c  p  s  cg  l  l  r   n  a  g   h  p  p   a  r  p  r   p  a  g   a  r  a v  y  y   e  m  l  d   t  r  l   p  g  h   g  q  p  v   p  e  l

 841 agtcgcgcgg catcttgctg gacggcacca cggccgacgg cacgcacccg cctgctagct  s  r  a   a  s  c   w  t  a  p   r  p  t   a  r  t   r  l  l  aa  v  a  r   h  l  a   g  r  h   h  g  r  r   h  a  p   a  c  - q  s  r   g  i  l  l   d  g  t   t  a  d   g  t  h  p   p  a  s

 901 tcagatcttc tccacgccca tgctgggccc ggtgttcttc gaattcatcc agcgcgaggg  s  d  l   l  h  a   h  a  g  p   g  v  l   r  i  h   p  a  r  gl  q  i  f   s  t  p   m  l  g   p  v  f  f   e  f i q r e f  r  s   s  p  r  p   c  w  a   r  c  s   s  n  s  s   s  a  r  961cgactaccgc gacggctttg gcgaaggcaa cttcaaggcg ctgttcgagt cgctggaacg  r  l  p   r  r  l   w  r  r  q   l  q  g   a  v  r   v  a  g  tg  d  y  r   d  g  f   g  e  g   n  f  k  a   l  f  e   s  l  e a  t  t   a  t  a  l   a  k  a   t  s  r   r  c  s  s   r  w  n 1021cgaccagatc cgccgtggtg tgctgaacac ataagacatc agacatccag ggttaaccct  r  p  d   p  p  w   c  a  e  h   i  r  h   q  t  s   r  v  n  pr  d  q  i   r  r  g   v  l  n   t  -  d  i   r  h  p   g  l  t a  t  r   s  a  v  v   c  -  t   h  k  t   s  d  i  q   g  -  p 1081gcacaggtgc ctatactgcg cgctccccgg aactcaaaag gatcccgatg tcgctccgta  a  q  v   p  i  l   r  a  p  r   n  s  k   g  s  r   c  r  s  vl  h  r  c   l  y  c   a  l  p   g  t  q  k   d  p  d   v  a  p c  t  g   a  y  t  a   r  s  p   e  l  k   r  i  p  m   s  l  r 1141gcaccctgtt cagcaccctt ttggccggcg cagccactgt cgcgctggcg cagaacccgt  a  p  c   s  a  p   f  w  p  a   q  p  l   s  r  w   r  r  t  r-  h  p  v   q  h  p   f  g  r   r  s  h  c   r  a  g   a  e  p s  t  l   f  s  t  l   l  a  g   a  a  t   v  a  l  a   q  n  p 1201ctgcccgctc acatcg (SEQ ID NO: 125)   l  p  a   h  i  (SEQ ID NO: 126)v  c  p  l   t  s (SEQ ID NO: 127)  s a  r  s  h     (SEQ ID NO: 128)

Sequence-tag of the clone M091_M24dG12. The primers 1j and 5h areindicated in the above sequence-tag of the clone M091_M24dG12.Hybridization between primer and template is indicated by arrows,mismatches by open circles. Putative conserved structural motifs aredepicted in bold and underlined.

A further sequence-tag (SP1_(—)2b5h) was generated using the primer pair2b/5h. A BlastX-analysis of the sequence from this tag yielded thehighest homology to an arylesterase from Agrobacterium tumefaciens, with70% identity. The single clone carrying the corresponding gene wasidentified (M4aE11) and the full length sequence determined to be asshown below:

1 atgaagacca ttctcgccta tggcgacagc ctgacctatg gggccaaccc gatcccgggc  m  k  t   i  l  a   y   g  d  s   l   t  y   g  a  n   p  i  p  g 61gggccgcggc atgcctatga ggatcgctgg cccacggcgc tggagcaggg gctgggcggc  g  p  r   h  a  y   e  d  r  w   p  t  a   l  e  q   g  l  g  g 121aaggcgcggg tgattgccga ggggctgggt ggtcgcacca cggtgcatga cgactggttt  k  a  r   v  i  a   e  g  l  g    g  r  t   t   v  h   d  d  w  f 181gcgaatgcgg acaggaacgg tgcgcgggtg ctgccgacgc tgctcgagag ccattcgccg  a  n  a   d  r  n   g  a  r  v   l  p  t   l  l  e   s  h  s  p 241ctcgacctga tcgtcatcat gctcggcacc aacgacatca agccgcatca cgggcggacg  l  d  l   i  v  i   m  l   g  t   n  d   i   k  p  h   h  g  r  t 301gccggcgagg ccgggcgggg catggcgcgg ctggtgcaga tcatccgcgg gcactatgcc  a  g  e   a  g  r   g  m  a  r   l  v  q   i  i  r   g  h  y  a 361ggccgcatgc aggacgagcc gcagatcatc ctcgtgtcgc cgccgccgat catcctcggc  g  r  m   q  d  e   p  q  i  i   l  v  s   p  p  p   i  i  l  g 421gactgggcgg acatgatgga ccatttcggc ccgcacgaag cgatcgccac ctcggtggat  d  w  a   d  m  m   d  h  f  g   p  h  e   a  i  a   t  s  v  d 481ttcgctcgcg agtacaagaa gcgggccgac gagcagaagg tgcatttctt cgacgccggc  f  a  r   e  y  k   k  r  a  d   e  q  k   v  h  f   f  d  a  g 541acggtggcga cgaccagcaa ggccgatggc atccacctcg acccggccaa tacgcgcgcc  t  v  a   t  t  s   k  a   d  g   i  h  l    d  p  a   n  t  r  a 601atcggggcag ggctggtgcc gctggtgaag caggtgctcg gcctgtaa (SEQ ID NO: 129)  i  g  a   g  l  v   p  l  v  k   q  v  l   g  l  — (SEQ ID NO: 130)

In the above sequence, the conserved structural motifs are shown in boldand underlined. The BlastP-analysis with the deduced full length aminoacid sequence identified the same hit with a identity of 48%. Theprimary structure of this enzyme showed the “GRTT”-motif proving theusefulness of the primers directed towards block 2 for theidentification of “GRTT”-esterases. The gene was amplified to introduceunique restriction enzyme recognition sites and the absence of secondsite mutations was confirmed by sequencing. The gene was ligated topET26 and was expressed in E. coli Rosetta (DE3). The vector map isprovided in FIG. 5. Expression and control strains were cultivated in LBin the presence of kanamycin (25 μg/ml), chloramphenicol (12.5 μg/ml),and 1% glucose. At an OD₅₈₀ of 1, expression was induced by addition of100 μM IPTG. Samples were taken at 2, 4, and 20 hours after induction.Cells were separated from the culture supernatant by centrifugation andafter resuspending in sample buffer, they wee incubated for 10 minutesat 90° C. An amount of cells representing an OD₅₈₀ of 0.1 was applied toa 4-12% acryl amide gradient gel, which was stained with CoomassieBrilliant Blue R250.

Strong overexpression of the gene was detected already 2 h afterinduction with 100 μM IPTG, as determined by SDS-PAGE analysis of crudecell extracts from E. coli Rosetta (DE3) pET26_M4aE11. The amount ofprotein representing M4aE11 (calculated size 23.2 kDa) increased furtherover time.

Esterase activity of crude cell extracts from strains expressing the“GDSL”-type esterase M4aE11 was determined. An amount of cellscorresponding to an O.D.₅₈₀=2 were resuspended in 200 μl of 5 mMTris/HCl pH 8.0, and lysed by ultrasonication. Then, 20 μl of eachsample were used to determine the esterase activity towardsp-nitrophenyl butyrate in a total volume of 200 μl. The activity wascorrected for the background activity of the control strain. Theactivity towards p-nitrophenylbutyrate reached about 125 nmol/ml×min 20h after induction.

In addition, SDS-PAGE analysis of the soluble and insoluble fraction ofcrude cell extracts from E. coli Rosetta (DE3) pET26_M4aE11 wasconducted. Cells from a culture induced with 100 μM IPTG and harvested 4h and 20 h after induction were lysed by ultrasonication and separatedinto soluble and insoluble fraction by centrifugation. Sample buffer wasadded and directly comparable amounts of soluble and insoluble fractionswere applied to a 4-12% acryl amide gradient gel, which was stained withCoomassie Brilliant Blue R250. The results of this analysis of thesolubility revealed that M4aE11 is partially (estimated 80%) soluble.The screening of the library M091 was completed.

Thus, in total nine different “GDSL”-type esterases were identified in 6different large insert metagenomic libraries and the esterases/lipasesBRAIN's library comprising more than 4.3 Gbp. Eight of these genes wereheterologously expressed in E. coli. The resulting enzyme samples ofseven clones were characterized for the desired perhydrolase activity.Two of the enzymes displayed this activity. Table 3 summarizes thescreening, expression and characterization of the metagenomic“GDSL”-type esterases.

TABLE 3 Expression and Characterization of Metagenomic “GDSL”-TypeEsterases Per- GDSL-type Homol- Expression² Solu- Activ- hydrolaseEsterase ogy¹ Level bility³ ity⁴ Activity S248_M2bB11 12.9% ++ + 136 −S248_M40cD4 14.8% +++ ++ 50 −/+⁶ S248_M44aA5 12.4% +++ ++ 75 −/+S261_M2aA12 36.9% ++ ++ 72 +⁷ S279_M70aE8 11.9% +++ + 167 − S279_M75bA25.7% n.d⁵. n.d. n.d. n.d.⁵ M091_M4aE11 33.9% +++ ++ 125 n.d. Est105 4.3%+++ − − n.d. Est114 7.8% n.d. n.d. 13 − ¹identity to the prototypeenzyme from M. smegmatis calculated with the dialign algorithm(Morgenstern et al., 1996) ²expression level: + moderate overexpression;++ strong overexpression; +++ very ³strong overexpression as judged fromSDS-PAGE-analysis as judged by SDS-PAGE-analysis ⁴towards p-nitrophenylbutyrate; given as nmol/(ml × min) ⁵not determined ⁶perhydrolysisactivity 2x background ⁷perhydrolase activity more than 2x backgroundEngineering of the Perhydrolase

Based on the structure of the perhydrolase, residues which may altersubstrate specificity (e.g., Km, kcat, Vmax, chain length, etc.) and/orthe multimeric nature of the protein were identified. However, it is notintended that the present invention be limited to any particularresidues. Nonetheless, site saturation libraries of residues D10, L12,T13, W14, W16, S54, A55, N94, K97, Y99, P146, W149, F150, I194, F196,are constructed, as well as combinatorial libraries of residues: E51A,Y73A, H81D, T127Q and single mutations of the active site residuesD192A, H195A and a site saturation library of the conserved D95. Methodsfor production of such libraries are known to those skilled in the artand include commercially available kits as the Stratagene Quikchange™Site-directed mutagenesis kit and/or Quikchange™ Multi-Site-directedmutagenesis kit.

Perhydrolase Activity

The use of enzymes obtained from microorganisms is long-standing. Indeedthere are numerous biocatalysts known in the art. For example, U.S. Pat.No. 5,240,835 (herein incorporated by reference) provides a descriptionof the transacylase activity of obtained from C. oxydans and itsproduction. In addition, U.S. Pat. No. 3,823,070 (herein incorporated byreference) provides a description of a Corynebacterium that producescertain fatty acids from an n-paraffin. U.S. Pat. No. 4,594,324 (hereinincorporated by reference) provides a description of a Methylcoccuscapsulatus that oxidizes alkenes. Additional biocatalysts are known inthe art (See e.g., U.S. Pat. Nos. 4,008,125 and 4,415,657; both of whichare herein incorporated by reference). EP 0 280 232 describes the use ofa C. oxydans enzyme in a reaction between a diol and an ester of aceticacid to produce monoacetate. Additional references describe the use of aC. oxydans enzyme to make chiral hydroxycarboxylic acid from a prochiraldiol. Additional details regarding the activity of the C. oxydanstransacylase as well as the culture of C. oxydans, preparation andpurification of the enzyme are provided by U.S. Pat. No. 5,240,835(incorporated by reference, as indicated above). Thus, thetransesterification capabilities of this enzyme, using mostly aceticacid esters were known. However, the determination that this enzymecould carry out perhydrolysis reaction was quite unexpected. It was evenmore surprising that these enzymes exhibit very high efficiencies inperhydrolysis reactions. For example, in the presence of tributyrin andwater, the enzyme acts to produce butyric acid, while in the presence oftributyrin, water and hydrogen peroxide, the enzyme acts to producemostly peracetic acid and very little butyric acid. This highperhydrolysis to hydrolysis ratio is a unique property exhibited by theperhydrolase class of enzymes of the present invention and is a uniquecharacteristic that is not exhibited by previously described lipases,cutinases, nor esterases.

The perhydrolase of the present invention is active over a wide pH andtemperature range and accepts a wide range of substrates for acyltransfer. Acceptors include water (hydrolysis), hydrogen peroxide(perhydrolysis) and alcohols (classical acyl transfer). Forperhydrolysis measurements, enzyme is incubated in a buffer of choice ata specified temperature with a substrate ester in the presence ofhydrogen peroxide. Typical substrates used to measure perhydrolysisinclude esters such as ethyl acetate, triacetin, tributyrin, ethoxylatedneodol acetate esters, and others. In addition, the wild type enzymehydrolyzes nitrophenylesters of short chain acids. The latter areconvenient substrates to measure enzyme concentration. Peracid andacetic acid can be measured by the assays described herein.Nitrophenylester hydrolysis is also described.

Although the primary example used during the development of the presentinvention is the M. smegmatis perhydrolase, any perhydrolase obtainedfrom any source which converts the ester into mostly peracids in thepresence of hydrogen peroxide finds use in the present invention.

Substrates

In some preferred embodiments of the present invention, esterscomprising aliphatic and/or aromatic carboxylic acids and alcohols areutilized with the perhydrolase enzymes of the present invention. In somepreferred embodiments, the substrates are selected from one or more ofthe following: formic acid, acetic acid, propionic acid, butyric acid,valeric acid, caproic acid, caprylic acid, nonanoic acid, decanoic acid,dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleicacid. In additional embodiments, triacetin, tributyrin, neodol esters,and/or ethoxylated neodol esters serve as acyl donors for peracidformation.

Cleaning and Detergent Formulations

The detergent compositions of the present invention are provided in anysuitable form, including for example, as a liquid diluent, in granules,in emulsions, in gels, and pastes. When a solid detergent composition isemployed, the detergent is preferably formulated as granules.Preferably, the granules are formulated to additionally contain aprotecting agent (See e.g., U.S. application Ser. No. 07/642,669 filedJan. 17, 1991, incorporated herein by reference). Likewise, in someembodiments, the granules are formulated so as to contain materials toreduce the rate of dissolution of the granule into the wash medium (Seee.g., U.S. Pat. No. 5,254,283, incorporated herein by reference in itsentirety). In addition, the perhydrolase enzymes of the presentinvention find use in formulations in which substrate and enzyme arepresent in the same granule. Thus, in some embodiments, the efficacy ofthe enzyme is increased by the provision of high local concentrations ofenzyme and substrate (See e.g., U.S. Patent Application PublicationUS2003/0191033, herein incorporated by reference).

Many of the protein variants of the present invention are useful informulating various detergent compositions. A number of known compoundsare suitable surfactants useful in compositions comprising the proteinmutants of the invention. These include nonionic, anionic, cationic,anionic or zwitterionic detergents (See e.g., U.S. Pat. Nos. 4,404,128and 4,261,868). A suitable detergent formulation is that described inU.S. Pat. No. 5,204,015 (previously incorporated by reference). Those inthe art are familiar with the different formulations which find use ascleaning compositions. As indicated above, in some preferredembodiments, the detergent compositions of the present invention employa surface active agent (i.e., surfactant) including anionic, non-ionicand ampholytic surfactants well known for their use in detergentcompositions. Some surfactants suitable for use in the present inventionare described in British Patent Application No. 2 094 826 A,incorporated herein by reference. In some embodiments, mixturessurfactants are used in the present invention.

Suitable anionic surfactants for use in the detergent composition of thepresent invention include linear or branched alkylbenzene sulfonates;alkyl or alkenyl ether sulfates having linear or branched alkyl groupsor alkenyl groups; alkyl or alkenyl sulfates; olefin sulfonates; alkanesulfonates and the like. Suitable counter ions for anionic surfactantsinclude alkali metal ions such as sodium and potassium; alkaline earthmetal ions such as calcium and magnesium; ammonium ion; andalkanolamines having 1 to 3 alkanol groups of carbon number 2 or 3.

Ampholytic surfactants that find use in the present invention includequaternary ammonium salt sulfonates, betaine-type ampholyticsurfactants, and the like. Such ampholytic surfactants have both thepositive and negative charged groups in the same molecule.

Nonionic surfactants that find use in the present invention generallycomprise polyoxyalkylene ethers, as well as higher fatty acidalkanolamides or alkylene oxide adduct thereof, fatty acid glycerinemonoesters, and the like.

In some preferred embodiments, the surfactant or surfactant mixtureincluded in the detergent compositions of the present invention isprovided in an amount from about 1 weight percent to about 95 weightpercent of the total detergent composition and preferably from about 5weight percent to about 45 weight percent of the total detergentcomposition. In various embodiments, numerous other components areincluded in the compositions of the present invention. Many of these aredescribed below. It is not intended that the present invention belimited to these specific examples. Indeed, it is contemplated thatadditional compounds will find use in the present invention. Thedescriptions below merely illustrate some optional components.

Proteins, particularly the perhydrolase of the present invention can beformulated into known powdered and liquid detergents having pH between 3and 12.0, at levels of about 0.001 to about 5% (preferably 0.1% to 0.5%)by weight. In some embodiments, these detergent cleaning compositionsfurther include other enzymes such as proteases, amylases, mannanases,peroxidases, oxido reductases, cellulases, lipases, cutinases,pectinases, pectin lyases, xylanases, and/or endoglycosidases, as wellas builders and stabilizers.

In addition to typical cleaning compositions, it is readily understoodthat perhydrolase variants of the present invention find use in anypurpose that the native or wild-type enzyme is used. Thus, such variantscan be used, for example, in bar and liquid soap applications, dishcareformulations, surface cleaning applications, contact lens cleaningsolutions or products, waste treatment, textile applications,pulp-bleaching, disinfectants, skin care, oral care, hair care, etc.Indeed, it is not intended that any variants of the perhydrolase of thepresent invention be limited to any particular use. For example, thevariant perhydrolases of the present invention may comprise, in additionto decreased allergenicity, enhanced performance in a detergentcomposition (as compared to the wild-type or unmodified perhydrolase).

The addition of proteins to conventional cleaning compositions does notcreate any special use limitations. In other words, any temperature andpH suitable for the detergent are also suitable for the presentcompositions, as long as the pH is within the range in which theenzyme(s) is/are active, and the temperature is below the describedprotein's denaturing temperature. In addition, proteins of the inventionfind use in cleaning, bleaching, and disinfecting compositions withoutdetergents, again either alone or in combination with a source ofhydrogen peroxide, an ester substrate (e.g., either added or inherent inthe system utilized, such as with stains that contain esters, pulp thatcontains esters etc), other enzymes, surfactants, builders, stabilizers,etc. Indeed it is not intended that the present invention be limited toany particular formulation or application.

Substrates

In some preferred embodiments of the present invention, esterscomprising aliphatic and/or aromatic carboxylic acids and alcohols areutilized with the perhydrolase enzymes in the detergent formulations ofthe present invention. In some preferred embodiments, the substrates areselected from one or more of the following: formic acid, acetic acid,propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid,nonanoic acid, decanoic acid, dodecanoic acid, myristic acid, palmiticacid, stearic acid, and oleic acid. Thus, in some preferred embodiments,detergents comprising at least one perhydrolase, at least one hydrogenperoxide source, and at least one ester acid are provided.

Hydrolases

In addition to the perhydrolase described herein, various hydrolasesfind use in the present invention, including but not limited tocarboxylate ester hydrolase, thioester hydrolase, phosphate monoesterhydrolase, and phosphate diester hydrolase which act on ester bonds; athioether hydrolase which acts on ether bonds; and α-amino-acyl-peptidehydrolase, peptidyl-amino acid hydrolase, acyl-amino acid hydrolase,dipeptide hydrolase, and peptidyl-peptide hydrolase which act on peptidebonds, all these enzymes having high perhydrolysis to hydrolysis ratios(e.g., >1). Preferable among them are carboxylate ester hydrolase, andpeptidyl-peptide hydrolase. Suitable hydrolases include: (1) proteasesbelonging to the peptidyl-peptide hydrolase class (e.g., pepsin, pepsinB, rennin, trypsin, chymotrypsin A, chymotrypsin B, elastase,enterokinase, cathepsin C, papain, chymopapain, ficin, thrombin,fibrinolysin, renin, subtilisin, aspergillopeptidase A, collagenase,clostridiopeptidase B, kallikrein, gastrisin, cathepsin D, bromelin,keratinase, chymotrypsin C, pepsin C, aspergillopeptidase B, urokinase,carboxypeptidase A and B, and aminopeptidase); (2) carboxylate esterhydrolase including carboxyl esterase, lipase, pectin esterase, andchlorophyllase; and (3) enzymes having high perhydrolysis to hydrolysisratios. Especially effective among them are lipases, as well asesterases that exhibit high perhydrolysis to hydrolysis ratios, as wellas protein engineered esterases, cutinases, and lipases, using theprimary, secondary, tertiary, and/or quaternary structural features ofthe perhydrolases of the present invention.

The hydrolase is incorporated into the detergent composition as much asrequired according to the purpose. It should preferably be incorporatedin an amount of 0.0001 to 5 weight percent, and more preferably 0.02 to3 weight percent. This enzyme should be used in the form of granulesmade of crude enzyme alone or in combination with other enzymes and/orcomponents in the detergent composition. Granules of crude enzyme areused in such an amount that the purified enzyme is 0.001 to 50 weightpercent in the granules. The granules are used in an amount of 0.002 to20 and preferably 0.1 to 10 weight percent. In some embodiments, thegranules are formulated so as to contain an enzyme protecting agent anda dissolution retardant material (i.e., material that regulates thedissolution of granules during use).

Cationic Surfactants and Long-Chain Fatty Acid Salts

Such cationic surfactants and long-chain fatty acid salts includesaturated or fatty acid salts, alkyl or alkenyl ether carboxylic acidsalts, a-sulfofatty acid salts or esters, amino acid-type surfactants,phosphate ester surfactants, quaternary ammonium salts including thosehaving 3 to 4 alkyl substituents and up to 1 phenyl substituted alkylsubstituents. Suitable cationic surfactants and long-chain fatty acidsalts include those disclosed in British Patent Application No. 2 094826 A, the disclosure of which is incorporated herein by reference. Thecomposition may contain from about 1 to about 20 weight percent of suchcationic surfactants and long-chain fatty acid salts.

Builders

In some embodiments of the present invention, the composition containsfrom about 0 to about 50 weight percent of one or more buildercomponents selected from the group consisting of alkali metal salts andalkanolamine salts of the following compounds: phosphates, phosphonates,phosphonocarboxylates, salts of amino acids, aminopolyacetates highmolecular electrolytes, non-dissociating polymers, salts of dicarboxylicacids, and aluminosilicate salts. Examples of suitable divalentsequestering agents are disclosed in British Patent Application No. 2094 826 A, the disclosure of which is incorporated herein by reference.

In additional embodiments, compositions of the present invention containfrom about 1 to about 50 weight percent, preferably from about 5 toabout 30 weight percent, based on the composition of one or more alkalimetal salts of the following compounds as the alkalis or inorganicelectrolytes: silicates, carbonates and sulfates as well as organicalkalis such as triethanolamine, diethanolamine, monoethanolamine andtriisopropanolamine.

Anti-Redeposition Agents

In yet additional embodiments of the present invention, the compositionscontain from about 0.1 to about 5 weight percent of one or more of thefollowing compounds as antiredeposition agents: polyethylene glycol,polyvinyl alcohol, polyvinylpyrrolidone and carboxymethylcellulose. Insome preferred embodiments, a combination of carboxymethyl-celluloseand/or polyethylene glycol are utilized with the composition of thepresent invention as useful dirt removing compositions.

Bleaching Agents

The use of the perhydrolases of the present invention in combinationwith additional bleaching agent(s) such as sodium percarbonate, sodiumperborate, sodium sulfate/hydrogen peroxide adduct and sodiumchloride/hydrogen peroxide adduct and/or a photo-sensitive bleaching dyesuch as zinc or aluminum salt of sulfonated phthalocyanine furtherimproves the detergent effects. In additional embodiments, theperhydrolases of the present invention are used in combination withbleach boosters (e.g., TAED and/or NOBS).

Bluing Agents and Fluorescent Dyes

In some embodiments of the present invention, bluing agents andfluorescent dyes are incorporated in the composition. Examples ofsuitable bluing agents and fluorescent dyes are disclosed in BritishPatent Application No. 2 094 826 A, the disclosure of which isincorporated herein by reference.

Caking Inhibitors

In some embodiments of the present invention in which the composition ispowdered or solid, caking inhibitors are incorporated in thecomposition. Examples of suitable caking inhibitors includep-toluenesulfonic acid salts, xylenesulfonic acid salts, acetic acidsalts, sulfosuccinic acid salts, talc, finely pulverized silica, clay,calcium silicate (e.g., Micro-Cell by Johns Manville Co.), calciumcarbonate and magnesium oxide.

Antioxidants

The antioxidants include, for example, tert-butyl-hydroxytoluene,4,4′-butylidenebis(6-tert-butyl-3-methylphenol),2,2′-butylidenebis(6-tert-butyl-4-methylphenol), monostyrenated cresol,distyrenated cresol, monostyrenated phenol, distyrenated phenol and1,1-bis(4-hydroxy-phenyl)cyclohexane.

Solubilizers

In some embodiments, the compositions of the present invention alsoinclude solubilizers, including but not limited to lower alcohols (e.g.,ethanol, benzenesulfonate salts, and lower alkylbenzenesulfonate saltssuch as p-toluenesulfonate salts), glycols such as propylene glycol,acetylbenzene-sulfonate salts, acetamides, pyridinedicarboxylic acidamides, benzoate salts and urea.

In some embodiments, the detergent composition of the present inventionare used in a broad pH range of from acidic to alkaline pH. In apreferred embodiment, the detergent composition of the present inventionis used in mildly acidic, neutral or alkaline detergent wash mediahaving a pH of from above 4 to no more than about 12.

In addition to the ingredients described above, perfumes, buffers,preservatives, dyes and the like also find use with the presentinvention. These components are provided in concentrations and formsknown to those in the art.

In some embodiments, the powdered detergent bases of the presentinvention are prepared by any known preparation methods including aspray-drying method and a granulation method. The detergent baseobtained particularly by the spray-drying method and/or spray-dryinggranulation method are preferred. The detergent base obtained by thespray-drying method is not restricted with respect to preparationconditions. The detergent base obtained by the spray-drying method ishollow granules which are obtained by spraying an aqueous slurry ofheat-resistant ingredients, such as surface active agents and builders,into a hot space. After the spray-drying, perfumes, enzymes, bleachingagents, inorganic alkaline builders may be added. With a highly dense,granular detergent base obtained such as by the spray-drying-granulationmethod, various ingredients may also be added after the preparation ofthe base.

When the detergent base is a liquid, it may be either a homogeneoussolution or an inhomogeneous dispersion.

The detergent compositions of this invention may be incubated withfabric, for example soiled fabrics, in industrial and household uses attemperatures, reaction times and liquor ratios conventionally employedin these environments. The incubation conditions (i.e., the conditionseffective for treating materials with detergent compositions accordingto the present invention), are readily ascertainable by those of skillin the art. Accordingly, the appropriate conditions effective fortreatment with the present detergents correspond to those using similardetergent compositions which include wild-type perhydrolase.

As indicated above, detergents according to the present invention mayadditionally be formulated as a pre-wash in the appropriate solution atan intermediate pH where sufficient activity exists to provide desiredimprovements softening, depilling, pilling prevention, surface fiberremoval or cleaning. When the detergent composition is a pre-soak (e.g.,pre-wash or pre-treatment) composition, either as a liquid, spray, gelor paste composition, the perhydrolase enzyme is generally employed fromabout 0.00001% to about 5% weight percent based on the total weight ofthe pre-soak or pre-treatment composition. In such compositions, asurfactant may optionally be employed and when employed, is generallypresent at a concentration of from about 0.0005 to about 1 weightpercent based on the total weight of the pre-soak. The remainder of thecomposition comprises conventional components used in the pre-soak(e.g., diluent, buffers, other enzymes (proteases), etc.) at theirconventional concentrations.

Cleaning Compositions Comprising Perhydrolase

The cleaning compositions of the present invention may be advantageouslyemployed for example, in laundry applications, hard surface cleaning,automatic dishwashing applications, as well as cosmetic applicationssuch as dentures, teeth, hair and skin. However, due to the uniqueadvantages of increased effectiveness in lower temperature solutions andthe superior color-safety profile, the enzymes of the present inventionare ideally suited for laundry applications such as the bleaching offabrics. Furthermore, the enzymes of the present invention find use inboth granular and liquid compositions.

The enzymes of the present invention also find use in cleaning additiveproducts. Cleaning additive products including the enzymes of thepresent invention are ideally suited for inclusion in wash processeswhere additional bleaching effectiveness is desired. Such instancesinclude, but are not limited to low temperature solution cleaningapplications. The additive product may be, in its simplest form, one ormore of the enzymes of the present invention. Such additive may bepackaged in dosage form for addition to a cleaning process where asource of peroxygen is employed and increased bleaching effectiveness isdesired. Such single dosage form may comprise a pill, tablet, gelcap orother single dosage unit such as pre-measured powders or liquids. Afiller or carrier material may be included to increase the volume ofsuch composition. Suitable filler or carrier materials include, but arenot limited to, various salts of sulfate, carbonate and silicate as wellas talc, clay and the like. Filler or carrier materials for liquidcompositions may be water or low molecular weight primary and secondaryalcohols including polyols and diols. Examples of such alcohols include,but are not limited to, methanol, ethanol, propanol and isopropanol. Thecompositions may contain from about 5% to about 90% of such materials.Acidic fillers can be used to reduce pH. Alternatively, the cleaningadditive may include activated peroxygen source defined below or theadjunct ingredients as defined below.

The cleaning compositions and cleaning additives of the presentinvention require an effective amount of the enzymes provided by thepresent invention. The required level of enzyme may be achieved by theaddition of one or more species of the M. smegmatis perhydrolase,variants, homologues, and/or other enzymes or enzyme fragments havingthe activity of the enzymes of the present invention. Typically, thecleaning compositions of the present invention comprise at least 0.0001weight percent, from about 0.0001 to about 1, from about 0.001 to about0.5, or even from about 0.01 to about 0.1 weight percent of at least oneenzyme of the present invention.

In some embodiments, the cleaning compositions of the present inventioncomprise a material selected from the group consisting of a peroxygensource, hydrogen peroxide and mixtures thereof, said peroxygen sourcebeing selected from the group consisting of:

(i) from about 0.01 to about 50, from about 0.1 to about 20, or evenfrom about 1 to 10 weight percent of a per-salt, an organic peroxyacid,urea hydrogen peroxide and mixtures thereof;

(ii) from about 0.01 to about 50, from about 0.1 to about 20, or evenfrom about 1 to 10 weight percent of a carbohydrate and from about0.0001 to about 1, from about 0.001 to about 0.5, from about 0.01 toabout 0.1 weight percent carbohydrate oxidase; and

(iii) mixtures thereof.

Suitable per-salts include those selected from the group consisting ofalkalimetal perborate, alkalimetal percarbonate, alkalimetalperphosphates, alkalimetal persulphates and mixtures thereof.

The carbohydrate may be selected from the group consisting ofmono-carbohydrates, di-carbohydrates, tri-carbohydrates,oligo-carbohydrates and mixtures thereof. Suitable carbohydrates includecarbohydrates selected from the group consisting of D-arabinose,L-arabinose, D-Cellobiose, 2-Deoxy-D-galactose, 2-Deoxy-D-ribose,D-Fructose, L-Fucose, D-Galactose, D-glucose, D-glycero-D-gulo-heptose,D-lactose, D-Lyxose, L-Lyxose, D-Maltose, D-Mannose, Melezitose,L-Melibiose, Palatinose, D-Raffinose, L-Rhamnose, D-Ribose, L-Sorbose,Stachyose, Sucrose, D-Trehalose, D-Xylose, L-Xylose and mixturesthereof.

Suitable carbohydrate oxidases include carbohydrate oxidases selectedfrom the group consisting of aldose oxidase (IUPAC classificationEC1.1.3.9), galactose oxidase (IUPAC classification EC1.1.3.9),cellobiose oxidase (IUPAC classification EC1.1.3.25), pyranose oxidase(IUPAC classification EC1.1.3.10), sorbose oxidase (IUPAC classificationEC1.1.3.11) and/or hexose oxidase (IUPAC classification EC1.1.3.5),Glucose oxidase (IUPAC classification EC1.1.3.4) and mixtures thereof.

In some preferred embodiments, the cleaning compositions of the presentinvention also include from about 0.01 to about 99.9, from about 0.01 toabout 50, from about 0.1 to 20, or even from about 1 to about 15 weightpercent a molecule comprising an ester moiety. Suitable moleculescomprising an ester moiety may have the formula:R¹O_(x)[(R²)_(m)(R³)_(n)]_(p)

wherein R¹ is a moiety selected from the group consisting of H or asubstituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl,alkylaryl, alkylheteroaryl, and heteroaryl; in one aspect of the presentinvention, R¹ may comprise from 1 to 50,000 carbon atoms, from 1 to10,000 carbon atoms, or even from 2 to 100 carbon atoms;

each R² is an alkoxylate moiety, in one aspect of the present invention,each R² is independently an ethoxylate, propoxylate or butoxylatemoiety;

R³ is an ester-forming moiety having the formula:

-   -   R⁴CO— wherein R⁴ may be H, substituted or unsubstituted alkyl,        alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and        heteroaryl, in one aspect of the present invention, R⁴ may be        substituted or unsubstituted alkyl, alkenyl, alkynyl, moiety        comprising from 1 to 22 carbon atoms, an aryl, alkylaryl,        alkylheteroaryl, or heteroaryl moiety comprising from 4 to 22        carbon atoms or R⁴ may be a substituted or unsubstituted C₁-C₂₂        alkyl moiety or R⁴ may be a substituted or unsubstituted C₁-C₁₂        alkyl moiety;    -   x is 1 when R¹ is H; when R¹ is not H, x is an integer that is        equal to or less than the number of carbons in R¹    -   p is an integer that is equal to or less than x    -   m is an integer from 0 to 50, an integer from 0 to 18, or an        integer from 0 to 12, and n is at least 1.

In one aspect of the present invention, the molecule comprising an estermoiety is an alkyl ethoxylate or propoxylate having the formulaR¹O_(x)[(R²)_(m)(R³)_(n)]_(p) wherein:

-   -   R¹ is an C₂-C₃₂ substituted or unsubstituted alkyl or        heteroalkyl moiety;    -   each R² is independently an ethoxylate or propoxylate moiety;    -   R³ is an ester-forming moiety having the formula:    -   R⁴CO— wherein R⁴ may be H, substituted or unsubstituted alkyl,        alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and        heteroaryl, in one aspect of the present invention, R⁴ may be a        substituted or unsubstituted alkyl, alkenyl, or alkynyl moiety        comprising from 1 to 22 carbon atoms, a substituted or        unsubstituted aryl, alkylaryl, alkylheteroaryl, or heteroaryl        moiety comprising from 4 to 22 carbon atoms or R⁴ may be a        substituted or unsubstituted C₁-C₂₂ alkyl moiety or R⁴ may be a        substituted or unsubstituted C₁-C₁₂ alkyl moiety;    -   x is an integer that is equal to or less than the number of        carbons in R¹    -   p is an integer that is equal to or less than x    -   m is an integer from 1 to 12, and    -   n is at least 1.

In one aspect of the present invention, the molecule comprising theester moiety has the formula:R¹O_(x)[(R²)_(m)(R³)_(n)]_(p)

wherein R¹ is H or a moiety that comprises a primary, secondary,tertiary or quaternary amine moiety, said R¹ moiety that comprises anamine moiety being selected from the group consisting of a substitutedor unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl,alkylheteroaryl, and heteroaryl; in one aspect of Applicants' inventionR¹ may comprise from 1 to 50,000 carbon atoms, from 1 to 10,000 carbonatoms, or even from 2 to 100 carbon atoms;

each R² is an alkoxylate moiety, in one aspect of the present inventioneach R² is independently an ethoxylate, propoxylate or butoxylatemoiety;

-   -   R³ is an ester-forming moiety having the formula:        -   R⁴CO— wherein R⁴ may be H, substituted or unsubstituted            alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl,            and heteroaryl, in one aspect of the present invention, R⁴            may be a substituted or unsubstituted alkyl, alkenyl, or            alkynyl moiety comprising from 1 to 22 carbon atoms, a            substituted or unsubstituted aryl, alkylaryl,            alkylheteroaryl, or heteroaryl moiety comprising from 4 to            22 carbon atoms or R⁴ may be a substituted or unsubstituted            C₁-C₂₂ alkyl moiety or R⁴ may be a substituted or            unsubstituted C₁-C₁₂ alkyl moiety;    -   x is 1 when R¹ is H; when R¹ is not H, x is an integer that is        equal to or less than the number of carbons in R¹    -   p is an integer that is equal to or less than x    -   m is an integer from 0 to 12 or even 1 to 12, and    -   n is at least 1.

In any of the aforementioned aspects of the present invention, themolecule comprising an ester moiety may have a weight average molecularweight of less than 600,000 Daltons, less than 300,000 Daltons, lessthan 100,000 Daltons or even less than 60,000 Daltons.

Suitable molecules that comprise an ester moiety includepolycarbohydrates that comprise an ester moiety.

The cleaning compositions provided herein will typically be formulatedsuch that, during use in aqueous cleaning operations, the wash waterwill have a pH of from about 5.0 to about 11.5, or even from about 7.5to about 10.5. Liquid product formulations are typically formulated tohave a pH from about 3.0 and about 9.0. Granular laundry products aretypically formulated to have a pH from about 9 to about 11. Techniquesfor controlling pH at recommended usage levels include the use ofbuffers, alkalis, acids, etc., and are well known to those skilled inthe art.

When the enzyme(s) of the present invention is/are employed in agranular composition or liquid, it may be desirable for the enzyme(s) tobe in the form of an encapsulated particle to protect such enzyme fromother components of the granular composition during storage. Inaddition, encapsulation is also a means of controlling the availabilityof the enzyme(s) during the cleaning process and may enhance performanceof the enzyme(s). In this regard, the enzyme(s) may be encapsulated withany encapsulating material known in the art.

The encapsulating material typically encapsulates at least part of theenzyme(s). Typically, the encapsulating material is water-soluble and/orwater-dispersible. The encapsulating material may have a glasstransition temperature (Tg) of 0° C. or higher. Glass transitiontemperature is described in more detail in WO 97/1151, especially frompage 6, line 25 to page 7, line 2.

The encapsulating material may be selected from the group consisting ofcarbohydrates, natural or synthetic gums, chitin and chitosan, celluloseand cellulose derivatives, silicates, phosphates, borates, polyvinylalcohol, polyethylene glycol, paraffin waxes and combinations thereof.When the encapsulating material is a carbohydrate, it may be typicallyselected from the group consisting of monosaccharides, oligosaccharides,polysaccharides, and combinations thereof. Typically, the encapsulatingmaterial is a starch. Suitable starches are described in EP 0 922 499;U.S. Pat. No. 4,977,252; U.S. Pat. No. 5,354,559 and U.S. Pat. No.5,935,826.

The encapsulating material may be a microsphere made from plastic suchas thermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile,polymethacrylonitrile and mixtures thereof; commercially availablemicrospheres that can be used are those supplied by Expancel ofStockviksverken, Sweden under the trademark EXPANCEL®, and thosesupplied by PQ Corp. of Valley Forge, Pa. U.S.A. under the tradename PM6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES®, LUXSIL®, Q-CEL® andSPHERICEL®.

Processes of Making and Using the Cleaning Compositions of the PresentInvention

The cleaning compositions of the present invention can be formulatedinto any suitable form and prepared by any process chosen by theformulator, non-limiting examples of which are described in U.S. Pat.No. 5,879,584; U.S. Pat. No. 5,691,297; U.S. Pat. No. 5,574,005; U.S.Pat. No. 5,569,645; U.S. Pat. No. 5,565,422 Del Greco et al.; U.S. Pat.No. 5,516,448; U.S. Pat. No. 5,489,392; and U.S. Pat. No. 5,486,303; allof which are incorporated herein by reference.

Adjunct Materials in Addition to the Enzymes of the Present Invention,Hydrogen Peroxide, and/or Hydrogen Peroxide Source and MaterialComprising an Ester Moiety

While not essential for the purposes of the present invention, thenon-limiting list of adjuncts illustrated hereinafter are suitable foruse in the instant cleaning compositions and may be desirablyincorporated in certain embodiments of the invention, for example toassist or enhance cleaning performance, for treatment of the substrateto be cleaned, or to modify the aesthetics of the cleaning compositionas is the case with perfumes, colorants, dyes or the like. It isunderstood that such adjuncts are in addition to the enzymes of thepresent invention, hydrogen peroxide and/or hydrogen peroxide source andmaterial comprising an ester moiety. The precise nature of theseadditional components, and levels of incorporation thereof, will dependon the physical form of the composition and the nature of the cleaningoperation for which it is to be used. Suitable adjunct materialsinclude, but are not limited to, surfactants, builders, chelatingagents, dye transfer inhibiting agents, deposition aids, dispersants,additional enzymes, and enzyme stabilizers, catalytic materials, bleachactivators, bleach boosters, preformed peracids, polymeric dispersingagents, clay soil removal/anti-redeposition agents, brighteners, sudssuppressors, dyes, perfumes, structure elasticizing agents, fabricsofteners, carriers, hydrotropes, processing aids and/or pigments. Inaddition to the disclosure below, suitable examples of such otheradjuncts and levels of use are found in U.S. Pat. Nos. 5,576,282,6,306,812, and 6,326,348, herein incorporated by reference. Theaforementioned adjunct ingredients may constitute the balance of thecleaning compositions of the present invention.

Surfactants—The cleaning compositions according to the present inventionmay comprise a surfactant or surfactant system wherein the surfactantcan be selected from nonionic surfactants, anionic surfactants, cationicsurfactants, ampholytic surfactants, zwitterionic surfactants,semi-polar nonionic surfactants and mixtures thereof.

The surfactant is typically present at a level of from about 0.1% toabout 60%, from about 1% to about 50% or even from about 5% to about 40%by weight of the subject cleaning composition.

Builders—The cleaning compositions of the present invention may compriseone or more detergent builders or builder systems. When a builder isused, the subject cleaning composition will typically comprise at leastabout 1%, from about 3% to about 60% or even from about 5% to about 40%builder by weight of the subject cleaning composition.

Builders include, but are not limited to, the alkali metal, ammonium andalkanolammonium salts of polyphosphates, alkali metal silicates,alkaline earth and alkali metal carbonates, aluminosilicate builderspolycarboxylate compounds. ether hydroxypolycarboxylates, copolymers ofmaleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxybenzene-2,4,6-trisulphonic acid, and carboxymethyloxysuccinic acid, thevarious alkali metal, ammonium and substituted ammonium salts ofpolyacetic acids such as ethylenediamine tetraacetic acid andnitrilotriacetic acid, as well as polycarboxylates such as melliticacid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid,benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, andsoluble salts thereof.

Chelating Agents—The cleaning compositions herein may contain achelating agent, Suitable chelating agents include copper, iron and/ormanganese chelating agents and mixtures thereof.

When a chelating agent is used, the cleaning composition may comprisefrom about 0.1% to about 15% or even from about 3.0% to about 10%chelating agent by weight of the subject cleaning composition.

Deposition Aid—The cleaning compositions herein may contain a depositionaid. Suitable deposition aids include, polyethylene glycol,polypropylene glycol, polycarboxylate, soil release polymers such aspolytelephthalic acid, clays such as Kaolinite, montmorillonite,atapulgite, illite, bentonite, halloysite, and mixtures thereof.

Dye Transfer Inhibiting Agents—The cleaning compositions of the presentinvention may also include one or more dye transfer inhibiting agents.Suitable polymeric dye transfer inhibiting agents include, but are notlimited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers,copolymers of N-vinylpyrrolidone and N-vinylimidazole,polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof.

When present in a subject cleaning composition, the dye transferinhibiting agents may be present at levels from about 0.0001% to about10%, from about 0.01% to about 5% or even from about 0.1% to about 3% byweight of the cleaning composition.

Dispersants—The cleaning compositions of the present invention can alsocontain dispersants. Suitable water-soluble organic materials includethe homo- or co-polymeric acids or their salts, in which thepolycarboxylic acid comprises at least two carboxyl radicals separatedfrom each other by not more than two carbon atoms.

Enzymes—The cleaning compositions can comprise one or more detergentenzymes which provide cleaning performance and/or fabric care benefits.Examples of suitable enzymes include, but are not limited to,hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases,phospholipases, esterases, cutinases, pectinases, keratinases,reductases, oxidases, phenoloxidases, lipoxygenases, ligninases,pullulanases, tannases, pentosanases, malanases, β-glucanases,arabinosidases, hyaluronidase, chondroitinase, laccase, and amylases, ormixtures thereof. A typical combination is cocktail of conventionalapplicable enzymes like protease, lipase, cutinase and/or cellulase inconjunction with amylase.

Enzyme Stabilizers—Enzymes for use in detergents can be stabilized byvarious techniques. The enzymes employed herein can be stabilized by thepresence of water-soluble sources of calcium and/or magnesium ions inthe finished compositions that provide such ions to the enzymes.

Catalytic Metal Complexes—The cleaning compositions of the presentinvention may include catalytic metal complexes. One type ofmetal-containing bleach catalyst is a catalyst system comprising atransition metal cation of defined bleach catalytic activity, such ascopper, iron, titanium, ruthenium, tungsten, molybdenum, or manganesecations, an auxiliary metal cation having little or no bleach catalyticactivity, such as zinc or aluminum cations, and a sequestrate havingdefined stability constants for the catalytic and auxiliary metalcations, particularly ethylenediaminetetraacetic acid,ethylenediaminetetra (methylenephosphonic acid) and water-soluble saltsthereof. Such catalysts are disclosed in U.S. Pat. No. 4,430,243.

If desired, the compositions herein can be catalyzed by means of amanganese compound. Such compounds and levels of use are well known inthe art and include, for example, the manganese-based catalystsdisclosed in U.S. Pat. No. 5,576,282.

Cobalt bleach catalysts useful herein are known, and are described, forexample, in U.S. Pat. No. 5,597,936; and U.S. Pat. No. 5,595,967. Suchcobalt catalysts are readily prepared by known procedures, such astaught for example in U.S. Pat. No. 5,597,936, and U.S. Pat. No.5,595,967.

Compositions herein may also suitably include a transition metal complexof a macropolycyclic rigid ligand—abreviated as “MRL”. As a practicalmatter, and not by way of limitation, the compositions and cleaningprocesses herein can be adjusted to provide on the order of at least onepart per hundred million of the active MRL species in the aqueouswashing medium, and will preferably provide from about 0.005 ppm toabout 25 ppm, more preferably from about 0.05 ppm to about 10 ppm, andmost preferably from about 0.1 ppm to about 5 ppm, of the MRL in thewash liquor.

Preferred transition-metals in the instant transition-metal bleachcatalyst include manganese, iron and chromium. Preferred MRL's hereinare a special type of ultra-rigid ligand that is cross-bridged such as5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane.

Suitable transition metal MRLs are readily prepared by known procedures,such as taught for example in WO 00/332601, and U.S. Pat. No. 6,225,464.

Method of Use

The cleaning compositions disclosed herein of can be used to clean asitus inter alia a surface or fabric. Typically at least a portion ofthe situs is contacted with an embodiment of Applicants' cleaningcomposition, in neat form or diluted in a wash liquor, and then thesitus is optionally washed and/or rinsed. For purposes of the presentinvention, washing includes but is not limited to, scrubbing, andmechanical agitation. The fabric may comprise most any fabric capable ofbeing laundered in normal consumer use conditions. The disclosedcleaning compositions are typically employed at concentrations of fromabout 500 ppm to about 15,000 ppm in solution. When the wash solvent iswater, the water temperature typically ranges from about 5° C. to about90° C. and, when the situs comprises a fabric, the water to fabric massratio is typically from about 1:1 to about 30:1.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: ° C. (degrees Centigrade); rpm (revolutions perminute); H₂O (water); HCl (hydrochloric acid); aa (amino acid); by (basepair); kb (kilobase pair); kD (kilodaltons); gm (grams); μg and ug(micrograms); mg (milligrams); ng (nanograms); μl and ul (microliters);ml (milliliters); mm (millimeters); nm (nanometers); μm and um(micrometer); M (molar); mM (millimolar); μM and uM (micromolar); U(units); V (volts); MW (molecular weight); sec (seconds); min(s)(minute/minutes); hr(s) (hour/hours); MgCl₂ (magnesium chloride); NaCl(sodium chloride); OD₂₈₀ (optical density at 280 nm); OD₆₀₀ (opticaldensity at 600 nm); PAGE (polyacrylamide gel electrophoresis); EtOH(ethanol); PBS (phosphate buffered saline [150 mM NaCl, 10 mM sodiumphosphate buffer, pH 7.2]); SDS (sodium dodecyl sulfate);Tris(tris(hydroxymethyl)aminomethane); TAED(N,N,N′N′-tetraacetylethylenediamine); w/v (weight to volume); v/v(volume to volume); Per (perhydrolase); per (perhydrolase gene); Ms (M.smegmatis); MS (mass spectroscopy); BRAIN (BRAIN Biotechnology Researchand Information Network, AG, Zwingenberg, Germany); TIGR (The Institutefor Genomic Research, Rockville, Md.); AATCC (American Association ofTextile and Coloring Chemists); WFK (wfk Testgewebe GmbH,Bruggen-Bracht, Germany); Amersham (Amersham Life Science, Inc.Arlington Heights, Ill.); ICN (ICN Pharmaceuticals, Inc., Costa Mesa,Calif.); Pierce (Pierce Biotechnology, Rockford, Ill.); Amicon (Amicon,Inc., Beverly, Mass.); ATCC (American Type Culture Collection, Manassas,Va.); Amersham (Amersham Biosciences, Inc., Piscataway, N.J.); BectonDickinson (Becton Dickinson Labware, Lincoln Park, N.J.); BioRad(BioRad, Richmond, Calif.); Clontech (CLONTECH Laboratories, Palo Alto,Calif.); Difco (Difco Laboratories, Detroit, Mich.); GIBCO BRL or GibcoBRL (Life Technologies, Inc., Gaithersburg, Md.); Novagen (Novagen,Inc., Madison, Wis.); Qiagen (Qiagen, Inc., Valencia, Calif.);Invitrogen (Invitrogen Corp., Carlsbad, Calif.); Genaissance(Genaissance Pharmaceuticals, Inc., New Haven, Conn.); DNA 2.0 (DNA 2.0,Menlo Park, Calif.); MIDI (MIDI Labs, Newark, Del.) InvivoGen(InvivoGen, San Diego, Calif.); Sigma (Sigma Chemical Co., St. Louis,Mo.); Sorvall (Sorvall Instruments, a subsidiary of DuPont Co.,Biotechnology Systems, Wilmington, Del.); Stratagene (Stratagene CloningSystems, La Jolla, Calif.); Roche (Hoffmann La Roche, Inc., Nutley,N.J.); Agilent (Agilent Technologies, Palo Alto, Calif.); Minolta(Konica Minolta, Ramsey, N.J.); and Zeiss (Carl Zeiss, Inc., Thornwood,N.Y.).

In the following Examples, various media were used. “TS” medium (perliter) was prepared using Tryptone (16 g) (Difco), Soytone (4 g)(Difco), Casein hydrolysate (20 g) (Sigma), K₂HPO₄ (10 g), and d H₂O (to1 L). The medium was sterilized by autoclaving. Then, sterile glucosewas added to 1.5% final concentration. Streptomyces Production Medium(per liter) was prepared using citric acid (H₂O) (2.4 g), Biospringeryeast extract (6 g), (NH₄)2SO₄ (2.4 g), MgSO₄.7H₂O (2.4 g), Mazu DF204(5 ml), trace elements (5 ml). The pH was adjusted to 6.9 with NaOH. Themedium was then autoclaved to sterilize. After sterilization, CaCl₂.2H₂O(2 mls of 100 mg/ml solution), KH₂PO₄ (200 ml of a 13% (w/v) solution atpH6.9), and 20 mls of a 50% glucose solution were added to the medium.

In these experiments, a spectrophotometer was used to measure theabsorbance of the products formed after the completion of the reactions.A reflectometer was used to measure the reflectance of the swatches.Unless otherwise indicated, protein concentrations were estimated byCoomassie Plus (Pierce), using BSA as the standard.

Example 1 Enzyme Analysis

In this Example, methods to assess enzyme purity and activity used inthe subsequent Examples and throughout the present Specification aredescribed.

Enzyme Activity Assay (pNB Assay)

This activity was measured by hydrolysis of p-nitrophenylbutyrate. Thereaction mixture was prepared by adding 10 ul of 100 mMp-nitrophenylbutyrate in dimethylsulfoxide to 990 ml of 100 mM Tris-HClbuffer, pH 8.0 containing 0.1% triton X-100. The background rate ofhydrolysis was measured before the addition of enzyme at 410 nm. Thereaction was initiated by the addition of 10 ul of enzyme to 990 ml ofthe reaction and the change of absorbance at 410 nm was measured at roomtemperate (˜23° C.). The background corrected results are reported asδA₄₁₀/min/ml or δA₄₁₀/min/mg protein.

Transesterification

Transesterification was measured by GC separation of products inbuffered aqueous reactions. Reactions to measure ethyl acetatetransesterification with propanol contained in 1 ml of 50 mM KPO4, pH7.0; 200 mM ethyl acetate, 200 mM 1-propanol, and enzyme. Reactions tomeasure ethyl acetate transesterification with neopentyl glycol (NPG)contained in 1 ml of 50 mM KPO4, pH 7.0; 303 mM ethyl acetate, 100 mMNPG, and enzyme. The reactions were incubated at the indicatedtemperatures and for the indicated times. Separations were preformedusing a 30M FFAP column (Phenomenex). The inlet split ratio wasapproximately 1:25, the injector was 250° C., head pressure of 10 psiHe, and detection was by FID at 250° C. The chromatography program was40° C. initial for 4 min, followed by a gradient of 15° C./min to 180°C. Components eluted in the following order and were not quantified;ethyl acetate, ethyl alcohol, propyl acetate, propyl alcohol, aceticacid, NPG diacetate, NPG monoacetate, and NPG.

Perhydrolase Used in Crystallography Studies

This perhydrolase preparation was used for crystallography studies. Inaddition, unlabelled protein was grown and purified in similar manner. A500 ml preculture of E. coli BL21(DE3)/pLysS/pMSATNco1-1 was grown in abaffled 2.8 L Fernbach flask on LB containing 100 ug/ml carbenicillin.After overnight culture at 37° C. and 200 rpm on a rotary shaker, thecells were harvested by centrifugation and resuspended in M9 mediumcontaining: glucose, 2 g/L; Na₂HPO₄, 6 g/L; KH₂PO₄, 3 g/L; NH₄Cl, 1 g/L;NaCl, 0.5 g/L; thiamine, 5 mg/L; MgSO₄, 2 mM; CaCl₂, 100 uM; Citricacid.H₂O, 40 mg/L; MnSO₄.H₂O, 30 mg/L; NaCl, 10 mg/L; FeSO₄.7H₂O, 1mg/L; CoCl₂.6H₂O, 1 mg/L; ZnSO₄.7H₂O, 1 mg/L; CuSO₄.5H₂O, 100 ug/L;H₃BO₃.5H₂O, 100 ug/L; and NaMoO₄.2H₂O, 100 ug/L; and supplemented withcarbenicillin, 100 mg/L. The resuspended cells were used to inoculatesix Fernbach flasks containing 500 ml each of M9 medium supplementedwith carbenicillin (100 mg/L). The cultures were incubated at 20° C. and200 rpm on a rotary shaker until the OD₆₀₀ reached about 0.7 at whichtime 100 mg/L of lysine, threonine, and phenylalanine and 50 mg/L ofleucine, isoleucine, valine, and selenomethionine were added. Afterfurther incubation for 30 min, IPTG was added to a final concentrationof 50 uM. The cultures were then incubated overnight (˜15 hr) andharvested by centrifugation. The cell pellet was washed 2 times with 50mM KPO₄ buffer, pH 6.8. The yield was 28.5 μm wet weight of cells towhich was added 114 ml of 100 mM KPO₄ buffer, pH 8.2 and 5 mg of DNase.This mixture was frozen at −80° C. and thawed 2 times.

The thawed cell suspension was lysed by disruption in a French pressurecell at 20K psi. The unbroken cells and cell membrane material weresedimented by centrifugation at 100K times g for 1 hour. The supernatantcrude extract, 128 ml (CE) was then placed in a 600 ml beaker andstirred for 10 minutes in a 55° C. water bath to precipitate unstableproteins. After 10 min the beaker was stirred in ice water for 1 minfollowed by centrifugation at 15K times g for 15 mM The supernatant fromthis procedure, HT, contained 118 ml. The HT extract was then made 20%saturating in (NH₄)₂SO₄ by the slow addition of 12.7 g of (NH₄)₂SO₄.This was loaded on to a 10 cm×11.6 cm Fast Flow Phenyl Sepharose(Pharmacia) column equilibrated in 100 mM KPO₄ buffer, pH 6.8,containing 20% saturation (109 g/L) (NH₄)₂SO₄. After loading the extractthe column was washed with 1700 ml of starting buffer and eluted with atwo step gradient. The first step was a linear 1900 ml gradient fromstart buffer to the same buffer without (NH₄)₂SO₄, the second was a 500ml elution with 100 mM KPO₄, pH 6.8 containing 5% EtOH. Activefractions, 241 ml, were pooled, diluted 100% with water and loaded ontoa 1.6 mm×16 mm Poros HQ strong anion exchange column equilibrated in 100mM Tris-HCl, pH 7.6. After loading the extract, the column was washedwith 5 column volumes of starting buffer. The protein was eluted with a15 column volume gradient from start buffer to start buffer containing175 mM KCl. The active fractions were pooled and concentrated using aCentriprep 30 (Millipore) to 740 μl. FIG. 6 provides a purificationtable showing the enzyme activity of the enzyme of the present inventionthrough various steps in the purification process.

The present application must be used to determine the respective valuesof the parameters of the present invention.

Unless otherwise noted, all component or composition levels are inreference to the active level of that component or composition, and areexclusive of impurities, for example, residual solvents or by-products,which may be present in commercially available sources.

Enzyme components weights provided herein are based on total activeprotein. All percentages and ratios were calculated by weight unlessotherwise indicated. All percentages and ratios were calculated based onthe total composition unless otherwise indicated.

Example 2 Determination of Ratio Between Peracid and Acid Formation

In this Example, methods for determining the ratio of perhydrolysis tohydrolysis are described. In particular, this Example provides methodsfor determining the ratio between peracid formation (i.e.,perhydrolysis) and acid formation (i.e., hydrolysis) resulting fromenzyme activity on an ester substrate in the presence of peroxide in anaqueous system.

A. Determination of Perhydrolysis to Hydrolysis Ratio

Preparation of Substrate

The substrates were prepared as described herein. Ethyl acetate (EtOAc)and other water soluble esters were diluted in a desired buffer to aconcentration of 10 mM of ester. Tributyrin and other water insolublesubstrates were prepared by making substrate swatches. Polyesterswatches were cut from non-dyed polyester fabric (Polycotton, PCW 22)using a ⅝ inch punch and placed in a 24-well microtiter plate (Costar,Cell Culture Plate). The insoluble ester was diluted to 1.03 M inhexane. Then, 10 μL of the insoluble ester solution were then adsorbedonto the polyester swatch.

Determination of Hydrolysis (GC Assay)

The hydrolytic assay described below was used to determine the amount ofsubstrate hydrolysis. In this assay, the assay solution was comprised of50 mM potassium phosphate pH 7.5, 10 mM ester substrate, 29 mM hydrogenperoxide, and 20 mM potassium chloride in a total volume of 0.99 ml andan amount of enzyme that would generate 20 nmoles of acetic acid perminute at 25° C.

For measuring water insoluble ester hydrolysis, the reaction mixture wasadded to the insoluble ester fabric swatch. The swatch was prepared asdescribed above (“Preparation of Substrate”). All the other conditionsfor the assay were the same except for exclusion of other estersubstrates.

Hydrolytic activity was measured by monitoring the increase of acidsgenerated by the enzyme from acyl donor substrates using gaschromatography coupled with flame ionization detection. The assay wasconducted by first pipetting 50 μL of assay solution containing all thecomponents except the enzyme into 200 mL of methanol (HPLC grade) todetermine the amount of acid in the assay solution at time 0. Then, 10μL of enzyme were added to the assay solution to a desired finalconcentration which produced approximately 20 nanomoles of acid perminute. A timer was started and 50 μL aliquots were taken from the assaysolution and added to 200 μL of methanol at various times, typically 2,5, 10, 15, 25, 40, and 60 minutes, after addition of the enzyme.

These methanol-quenched samples were then injected into a gaschromatograph coupled with a flame ionization detector (Agilent 6890N)and analyzed for hydrolytic components, acetic, and butyric acids. Gaschromatography was conducted using a nitroterephthalic acid modifiedpolyethylene glycol column (Zebron FFAP; with dimensions: 30 m long, 250um diameter, 250 nm film thickness). A 3 μL aliquot of sample wasapplied to the column by a splitless injection under constant a heliumflow of 1.0 mL/minute. The inlet was maintained at a temperature of 250°C. and was purged of any remaining sample components after 2 minutes.When analyzing acetic acid, the temperature of the column was maintainedat 75° C. for 1 minute after injection, increased 25° C./minute to 100°C., then increased 15° C./minute to 200° C.

When analyzing butyric acid, the temperature of the column wascontrolled as described above, except the temperature was additionallyincreased 25° C./minute to 225° C. and held at 225° C. for 1 minute. Theflame ionization detector was maintained throughout the chromatographyat 250° C. and under constant hydrogen flow of 25 mL/minute, air flow of200 mL/minute, and a combined column and makeup helium flow of 30mL/minute. The amount of hydrolyzed acid in the sample was thendetermined by integrating the acid peak in the chromatogram for totalion counts and calculating the acid from the ion count using a standardcurve generated under the above conditions for acetic and butyric acidsat varying concentrations in the assay solution (without enzyme).

Determination of Perhydrolysis (OPD Assay)

The perhydrolytic activity assay described below was used to determinethe amount of peracid formed in the reaction. In these assays, thesolution comprised 50 mM potassium phosphate pH 7.5, 10 mM estersubstrate, 29 mM hydrogen peroxide, 20 mM potassium chloride, and 10 mMO-phenylenediamine.

When using water insoluble ester as the acyl donor, an ester adsorbedfabric swatch was used as the substrate, prepared as described above(“Preparation of Substrate”).

Perhydrolytic activity was measured by monitoring the absorbanceincrease at 458 nm of oxidized O-phenylenediamine (OPD) by peracidgenerated with the enzyme. The perhydrolytic activity assay solution wasprepared in the same manner as the hydrolytic activity assay solution,except that OPD was added to the assay solution to a final concentrationof 10 mM. The OPD solution was prepared immediately before conductingthe assay by dissolving 72 mg OPD (Sigma-Aldrich, dihydrochloride) in19.94 mL of the same buffer and the pH was adjusted by slowly adding 60μL of 13.5 M potassium hydroxide. The pH was measured and if needed,small quantities of potassium hydroxide were added to return the pH tothe original pH of the buffer. Then, 495 μL of this OPD solution wereadded with the other assay components to a final assay volume of 0.990mL. An assay quenching solution was also prepared by dissolving 36 mgOPD in 20 mL 100 mM citric acid and 70% ethanol.

The assay was typically conducted at 25° C. The assay was started bypipetting 100 μL of assay solution before the addition of the enzymeinto 200 μL of quenching solution to determine the amount ofperhydrolytic components and background absorbance in the assay solutionat time 0. Then, 10 μL of enzyme were added to the assay solution to adesired final concentration which produced approximately 10 nanomoles ofperacid per minute. A timer was started and 100 μL aliquots were takenfrom the assay solution and added to 200 μL of quenching solution atvarious times, typically 2, 5, 10, 15, 25, 40, and 60 minutes, afteradding the enzyme. The quenched assay solutions were incubated for 30minutes to allow any remaining peracid to oxidize the OPD. Then, 100 μLof each quenched assay solution was transferred to a 96-well microtiterplate (Costar) and the absorbance of the solution was measured at 458 nmby a spectrophotometric plate reader (Molecular Devices, SpectraMAX250). The amount of peracid in each quenched sample was calculated usinga standard curve generated under the above conditions with peraceticacid at varying concentrations in the assay solution (without enzyme).

Perhydrolysis/Hydrolysis Ratio:Perhydrolysis/Hydrolysis ratio=Perhydrolysis measured in thePerhydrolysis assay/(Total acid detected in the hydrolysisassay−Perhydrolysis measured in the perhydrolysis assay)

The results of these experiments are provided in FIGS. 7, 10 and FIG.11. FIG. 7 provides a graph which shows the ratio of perbutyric acid tobutyric acid generated by various enzymes from 10 mM tributyrin and 29mM hydrogen peroxide in 40 minutes. FIG. 10 shows the ratio ofperbutyric acid to butyric acid generated by various enzymes from 10 mMtributyrin and 29 mM hydrogen peroxide in 4, 10, and 30 minutes. FIG. 11shows the ratio of peracetic acid to acetic acid generated by variousenzymes from 10 mM triacetin and 29 mM hydrogen peroxide in 4 and 10minutes. The results obtained in these experiments indicated that M.smegmatis perhydrolase homologues exhibited a ratio above 1 in theOPD/GC assays described above, while other classes of enzymes exhibitedratios significantly below 1.

Table 2-1 provides data showing the perhydrolysis activity of varioushomologues described herein on triacetin, as compared to the wild-typeM. smegmatis perhydrolase. The results provided in Table 2-2 indicatethat the perhydrolase has activity over a broad range of substrates. Inaddition to the results provided in these Tables, FIGS. 8 and 9 providedata showing that the perhydrolase of the present invention has broad pHand temperature range activities.

TABLE 2-1 Perhydrolysis Activity of Perhydrolase Homologues on Triacetinas Compared to M. smegmatis perhydrolase Perhydrolysis Ratio (homolog toExperiment Protein perhydrolase) A. pET26_Mlo 0.6 pET26b_Mbo 0.87pET26_SmeII 2.1 pET26b_Stm 0.17 pLO_SmeI 0.7 Perhydrolase 1.0000 Blank0.0660 B. pET26_S261_M2aA12 1.5 Perhydrolase 1 Blank 0.3 C. pet26 M40cD40.14 pet26 M44aA5 0.16 Perhydrolase 1 Blank 0.01

TABLE 2-2 Peracid Production by 1 ppm Wild-Type Perhydrolase with 29 mMH2O2 and Various Esters nmol Peracetic Acid/min 10 mM of 10 mM Esterwith of Ester on 0.5% 10 mM of Polycotton Ester Neodol Ester SwatchEthyl Acetate 5.00 Butyl Acetate 8.06 8.72 Hexyl Acetate 7.96 5.86 OctylAcetate 8.03 0.48 Ethyl Propionate 0.90 1.43 Butyl Propionate 2.47 3.39Hexyl Propionate 4.00 2.66 Isoamyl Acetate 7.83 17.69 CitronellylAcetate 7.25 4.27 Citronellyl 2.85 3.21 Propionate Dodecyl Acetate 3.950.19 Neodol 23-3 2.25 8.77 Acetate Neodol 23-6.5 2.73 10.12 AcetateNeodol 23-9 2.97 10.20 Acetate Ethylene Glycol 13.30 Diacetate PropyleneGlycol 13.17 Diacetate Triacetin 11.91 Tributyrin 0.66 2.70 Ethyl 0.49Methoxyacetate Linalyl Acetate 0.30 Ethyl Butyrate 0.31 EthylIsobutyrate 0.10 Ethyl 2- 0.11 methylbutyrate Ethyl Isovalerate 0.37Diethyl Maleate 0.75 Ethyl Glycolate 1.91B. Typical Perhydrolase Peracid Generation Assay:

Perhydrolase is active over a wide pH and temperature range and acceptsa wide range of substrates for acyl transfer. Acceptors include water(hydrolysis), hydrogen peroxide (perhydrolysis) and alcohols (classicalacyl transfer). For perhydrolysis measurements enzyme was incubated inthe buffer of choice at a specified temperature with a substrate esterin the presence of hydrogen peroxide. Typical substrates used to measureperhydrolysis include ethylacetate, triacetin, tributyrin, ethoxylatedneodol acetate esters, and others. In addition, the wild type enzyme wasfound able to hydrolyze nitrophenylesters of short chain acids. Thelatter are convenient substrates to measure enzyme concentration. Insome embodiments, peracid acid and acetic acid were measured by the ABTSor HPLC assays as described below. Nitrophenylester hydrolysis is alsodescribed below.

C. ABTS Assay (One Milliliter):

This assay provides a determination of peracetic acid produced byperhydrolase. This protocol was adapted from Karst et al., Analyst,122:567-571 [1997]). Briefly, a 100 μL aliquot of solution to beanalyzed was added to 1 mL 125 mM K⁺ citrate pH 5, 1 mM ABTS, 50 μM KI.Absorbance was measured at 420 nm for highest sensitivity. However,multiple additional wavelengths were sometimes used over the broadabsorption spectrum of ABTS. Calibration curves were constructed basedon known peracid concentration series.

D. HPLC (Model—Agilent 1100) Determination of Perhydrolase ReactionProducts:

For determination of the ratio of perhydrolysis to hydrolysis of theperhydrolase reaction, perhydrolase reaction samples were quenched byacidification to a final concentration of 0.24% methanesulfonic acid,and the products were separated by reverse phase HPLC on a Dionex OAcolumn (cat #062903; Dionex Corporation, Sunnyvale, Calif.). The mobilephase was 100 mM NaPO₄, pH 3.9 (buffer was prepared by titrating 100 mMNa₂PO₄ with methanesulfonic acid to pH 3.9) run under isocraticconditions at 30° C. Detection was at 210 nm Concentrations of productswere calculated by comparison of the integrated peak areas againstcalibration standards.

E. Nitrophenylester Hydrolysis Kinetic Assay

Enzyme and substrate were incubated in 100 mM Tris/HCl pH 8.0 (or 50 mMB(OH)₃ pH 9.5 or another buffer). Absorbance at 402 nm was monitored. Insome experiments, the assay was carried out in standard 1 mL cuvettes,while in other experiments, microtiter plate wells were used. The lattermethod was used for the screening of mutant libraries. Enzymeconcentration was determined by comparison to standard curves obtainedunder the same reaction conditions.

F. Para-Nitrophenylcaproate Hydrolysis Assay

The pNC6 substrate solution was prepared by mixing 1 mM pNC6 (100 mMstock solution), 1 ml DMSO, 19 mls 100 mM Phosphate (pH8), and glycerolto a final concentration of 10%. To assay samples, 10 μl of the celllysate were added to 190 μl of the substrate solution, and assayed at405 nm for 15 minutes in a spectrophotometer. The results are presentedas the average of two experiments.

G. Para-Nitrophenyl Acetate (pNA) Hydrolysis Assay

Aliquots of the lysed cell supernatant were diluted 1-100 in 100 mMphosphate buffer (pH 8). To assay the samples, 5 μl of the 1-100 dilutedcell supernatant were placed into each well of a microtiter plate. Then,195 μl of reaction buffer/substrate mix (1 mM pNA, 100 mM phosphate, pH8, 10% glycerol) were added, and the absorbance rate at 405 nm wasmeasured over 3 minutes (kinetics program, microtiter plate reader). Theresults are presented as the average of two experiments.

Example 3 Assays Including Detergent Compositions

In this Example, assay systems used to screen for superior perhydrolaseactivity in detergents with particular substrates are provided. Theseassays include those that measure peracid degradation of perhydrolase,as well as the peracid synthesis activity of the enzyme.

Materials and Methods for Peracetic Acid Formation (PAF) and PeraceticAcid Degradation (PAD) Assays

This section provides the materials and methods used to screen for asuperior perhydrolases in Aria with C9E2OAC ester substrate

Materials:

Aria Futur without bleach, perfume, or enzymes (P&G, Aria “C”)

C9E2OAc (P&G)

30% Hydrogen Peroxide (Sigma)

32% Peroxyacetic acid (“peracid”, PAA) (Sigma cat#) MW=76.05; 4.208M

Citric Acid, anhydrous MW=192.12

Potassium Hydroxide MW=56.11

ABTS (Sigma cat# A1888) MW=548.68

Potassium Iodide MW=166.0

Potassium Phosphate, mono and di-basic

Stock Solutions:

Ariel detergent stock: Aria Futur without bleach, perfume, or enzymes(“Aria C”) was dissolved in water to 6.72 g/L. It was stirred at roomtemp for 30 minutes, then allowed to settle. Then, it was divided intoconvenient aliquots and stored at 4° C., until used. When made and usedfresh, the solution was filtered, instead of settled100 mM C9E2OAc in Ariel detergent stock: First, 30 μl C9E2OAc was addedto 970 μl Aria detergent stock, using a positive displacement pipet. Itwas sonicated in a bath sonicator until a milky dispersion was formed(15-60 seconds). The dispersion was stable for about two hours. Whenused, 10 μl of dispersion per ml of reaction mix were used.42 mM Peroxyacetic acid stock: Right before use, the Sigma 32% PAAsolution was diluted 1:100 in water. Then 5.7 μl of the 42 mM stock perml of reaction mix was added.2 M hydrogen peroxide: One ml of 30% Sigma hydrogen peroxide was addedto 3.41 ml water. This solution was prepared fresh, right before use. Itwas used at 10 μl per ml of reaction mix.125 mM Citrate buffer pH 5.0: This was prepared to 24.0 grams per liter.It was made up in 800 ml, and titrated to pH 5.0 with 50% KOH. Thevolume was adjusted to 1 liter and stored at room temperature.100 mM ABTS stock: This was prepared using 549 mg of ABTS in 10 ml ofwater. It was frozen at −80° C., in convenient aliquots in opaqueEppendorf tubes. The stock was stable indefinitely when kept frozen inthe dark. ABTS will precipitate when thawed from −80° C. but goes backinto solution upon mixing. In use, 10 μl of ABTS stock was used per mlof ABTS reagent.250 mM KI: This was prepared as 415 mg in 10 ml water. It was kept at 4°C. It was diluted to 25 mM working stock, and 2 ul of working stock wasused per ml of ABTS reagent.25 mM Potassium Phosphate buffer, pH 8.0:Method:

The night prior to performance of the assays, the plates containinglysed cells that contain perhydrolase were checked to be sure that theywere frozen twice. On the day of the assay, 30 to 45 minutes wereallowed for the plates to thaw. The ABTS reagent was prepared and theMultidrop (Multidrop 384 instrument, ThermoElectron) to fill thedetection plates with 200 μl per well. Store the filled plates coveredat room temperature in the dark until needed. Dilutions of the standardswere prepared so that when 20 μl of the diluted standard were added tothe 180 μl of the reaction mix, the concentration in the well was 1 ppm.Four 4 two-fold serial dilutions were prepared to a set of sixstandards: 1, 0.5, 0.25, 0.125, and 0.0625 ppm final concentration inthe wells.

To test, 20 μl of the standards were added to the thawed 1:10 dilutionplate. The reaction mixtures were prepared and the Multidrop used tofill one reaction plate for each plate to be assayed (180 μl/well). Notethat the reaction mixtures are different for the PAF and PAD assays.

Peracid Hydrolysis (Peracid Degradation, PAD) Assay:

This assay measures the amount of peracetic acid remaining after a 100minute incubation with enzyme in an Aria detergent background. Theamount of peracid remaining is detected by reacting an aliquot of thereaction mixture with the ABTS detection reagent.

In this assay, 20 μl enzyme samples from the thawed 1:10 dilution platewere transferred, one column at a time with an 8 channel pipetter, intothe corresponding column of the pre-filled PAD reaction plate. A timerwas started as soon as transfer occurred from the first column;subsequent columns were transferred at 15 second intervals (i.e., thelast column was finished 2 min 45 sec. after starting the first one).The plate was mixed for 30 seconds on the thermomixer (750 rpm, to avoidsplashing). The plate was then transferred to a humidified chamber at25° C. The plate was incubated for a total of 100 minutes from the timethe first column of enzyme was added. At 100 minutes incubation, thereaction plate was removed from the incubator. Then, 20 ul aliquots ofthe reaction mixture were transferred to an ABTS reagent plate, in thesame order and with the same 15 second time interval that the enzymesamples were originally added to the reaction plate. The ABTS plate wasallowed to sit at room temperature for three minutes after the lastcolumn of reaction mixture was added. The plate was then read on thespectrophotometric plate reader at 420 and 740 nm.

Perhydrolysis (Peracid Formation, PAF) Assay

Multidrop Optimized Protocol: Screening for a Superior Perhydrolysis inAriel with C9E2OAC Ester Substrate

The same materials and stock solutions described above for PAD were usedin these experiments, as indicated below.

Method:

The methods were designed to assay 20 μl aliquots from a 1:100 dilutionplate. The 20 μl 1:100 dilution assay plates were produced during theprocess of obtaining the protein concentrations and were stored at −80°C. The plates were thawed for about 30 to 45 minutes before use.Dilutions of the S54V standards were prepared, so that when 2 μl of thediluted standard are added to the 20 μl of the 1:100 diluted celllysate, the concentration in the well was 0.1 ppm. Four two-fold serialdilutions were prepared to produced a set of six standards: 0.1, 0.05,0.025, 0.0125, and 0.00625 ppm final concentration in the wells. Then, 2ul of the standards were added to the thawed 20 ul 1:100 dilution assayplates in the wells indicated.

Perhydrolysis (Peracid Formation, PAF) Assay:

This assay measures the amount of peroxyacetic acid that is produced in10 minutes from the C9E2OAc substrate in an Aria detergent background.The amount of peracid formed is detected after 10 minutes by reacting analiquot of the reaction mixture with the ABTS detection reagent.

The Multidrop was used to deliver 180 μl/well of the PAF reaction mix tothe prepared 1:100 dilution plate. The timer was started and thereaction plate was placed on the thermomixer, with the temperature setat 25° C. The plate was covered and the solutions mixed for 30 secondsat 750 rpm. The plate was then allowed to rest on the thermomixerwithout mixing, for a total of 10 minutes from the time the reaction mixwas added. At 10 minutes, the Multidrop was used to add 20 μl/well ofthe 10×ABTS reagent. The 10× reagent was a milky suspension. Thethermomixer was used to briefly shake the plate. The ABTS reagentquickly went into solution. The plate was allowed to sit at roomtemperature for three minutes after the ABTS reagent was added. Theplate was then read on the spectrophotometric plate reader at 420 nm.

Example 4 Cloning of Mycobacterium smegmatis Perhydrolase

In this Example, the cloning of M. smegmatis perhydrolase is described.An enzyme with acyltransferase activity was purified fromCorynebacterium oxydans (now Mycobacterium parafortuitum ATCC19686). Twopeptide sequences were obtained from the purified protein. One peptidewas determined by Edman degradation from cyanogen bromide cleavage ofthe purified enzyme using methods known in the art. The sequence of thispeptide was determined to be KVPFFDAGSVISTDGVDGI (SEQ ID NO:3). Thesecond peptide was analyzed using N-terminal sequencing and was found tohave the GTRRILSFGDSLTWGWIPV (SEQ ID NO:4). A BLAST search against theTIGR unfinished genome database identified a sequence of potentialinterest in Mycobacterium smegmatis, which is shown below:

(SEQ ID NO: 2) MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTKAYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHF TEANNRDLGVALAEQVRSLL.

The corresponding DNA sequence of the gene is:

(SEQ ID NO: 1) 5′-ATGGCCAAGCGAATTCTGTGTTTCGGTGATTCCCTGACCTGGGGCTGGGTCCCCGTCGAAGACGGGGCACCCACCGAGCGGTTCGCCCCCGACGTGCGCTGGACCGGTGTGCTGGCCCAGCAGCTCGGAGCGGACTTCGAGGTGATCGAGGAGGGACTGAGCGCGCGCACCACCAACATCGACGACCCCACCGATCCGCGGCTCAACGGCGCGAGCTACCTGCCGTCGTGCCTCGCGACGCACCTGCCGCTCGACCTGGTGATCATCATGCTGGGCACCAACGACACCAAGGCCTACTTCCGGCGCACCCCGCTCGACATCGCGCTGGGCATGTCGGTGCTCGTCACGCAGGTGCTCACCAGCGCGGGCGGCGTCGGCACCACGTACCCGGCACCCAAGGTGCTGGTGGTCTCGCCGCCACCGCTGGCGCCCATGCCGCACCCCTGGTTCCAGTTGATCTTCGAGGGCGGCGAGCAGAAGACCACTGAGCTCGCCCGCGTGTACAGCGCGCTCGCGTCGTTCATGAAGGTGCCGTTCTTCGACGCGGGTTCGGTGATCAGCACCGACGGCGTCGACGGAATCCACTTCACCGAGGCCAACAATCGCGATCTCGGGGTGGCCCTCGCGGAACAGG TGCGGAGCCTGCTGTAA-3′

Primers were designed based on the gene sequence to amplify and clonethe gene. The primers used for amplification were:

MsRBSF: (SEQ ID NO: 5) 5′-CTAACAGGAGGAATTAACCATGGCCAAGCGAATTCTGTGTTTCGGTGATTCCCTGACCT-3′ MspetBamR: (SEQ ID NO: 6)5′-GCGCGCGGATCCGCGCGCTTACAGCAGGCTCCGCACCTGTTCCGCG AGGGCCACCCCGA-3′

The amplification of the gene was done by PCR using Taq DNA polymerase(Roche) per the manufacturer's instructions, with approximately 500 ngof chromosomal DNA from Mycobacterium smegmatis as the template DNA andthe addition of 1% DMSO to the PCR reaction mix. Thirty picomoles ofeach of the primers MsRBSF and MspetBamR were added to the mix. Theamplification cycle was: 30 cycles of (95° C. for 1 min, 55° C. for 1min, 72° C. for 1 min).

The fragments obtained from the PCR reaction were separated on a 1.2%agarose gel and a single band of the expected size of 651 bp (codingsequence and stop codon) was identified. This band was cloned directlyinto the pCR2.1 TOPO cloning vector (Invitrogen) and transformed into E.coli Top 10 cells (Invitrogen) with selection on L agar (10 g/ltryptone, 5 g/l yeast extract, 5 g/l NaCl, 20 g/l agar) containing 100micrograms/ml carbenicillin and X-gal (20 micrograms/ml, Sigma-Aldrich)for blue/white selection and incubated overnight at 37° C. Five whitecolonies were analyzed for the presence of the PCR fragment. Each colonywas used to inoculate 5 mls of L broth (L agar without the addition ofagar) containing 100 micrograms/ml carbenicillin and the cultures weregrown overnight at 37° C. with shaking at 200 rpm. Plasmid DNA waspurified from the cultures using the Quikspin kit (Qiagen). The presenceof the correct fragment was determined by restriction enzyme digest withEcoR1 to release the fragment, and sequencing using primers supplied bythe pCR2.1 manufacturer (Invitrogen). The correct plasmid was designatedpMSATNcoI (See, FIG. 12, for the map of this plasmid)). The sequence ofthis plasmid is provided below

(SEQ ID NO: 13) agcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagctatttaggtgacactatagaatactcaagctatgcatcaagcttggtaccgagctcggatccactagtaacggccgccagtgtgctggaattcgcccttctaacaggaggaattaaccatggccaagcgaattctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcacccaccgagcggttcgcccccgacgtgcgctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggagggactgagcgcgcgcaccaccaacatcgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgcgacgcacctgccgctcgacctggtgatcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgacatcgcgctgggcatgtcggtgctcgtcacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggcacccaaggtgctggtggtctcgccgccaccgctggcgcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccactgagctcgcccgcgtgtacagcgcgctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccgacggcgtcgacggaatccacttcaccgaggccaacaatcgcgatctcggggtggccctcgcggaacaggtgcagagcctgctgtaaaagggcgaattctgcagatatccatcacactggcggccgctcgagcatgcatctagagggcccaattcgccctatagtgagtcgtattacaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctatacgtacggcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgccggggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataaatgtcaggcatgagattatcaaaaaggatcttcacctagatccttttcacgtagaaagccagtccgcagaaacggtgctgaccccggatgaatgtcagctactgggctatctggacaagggaaaacgcaagcgcaaagagaaagcaggtagcttgcagtgggcttacatggcgatagctagactgggcggttttatggacagcaagcgaaccggaattgccagctggggcgccctctggtaaggttgggaagccctgcaaagtaaagctggatggctttctcgccgccaaggatctgatggcgcaggggatcaagctctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaattattaacgcttacaatttcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatagcacgtgaggagggccaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtgctaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctgggcttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagc gaggaagcggaagConstruction of Perhydrolase T7 Expression Plasmid

The primer pair used to create pMSATNco1 was also used to create an NcoIsite (CCATGG) in which the ATG is the start codon of the acyltransferasegene and a BamH1 (GGATCC) just after the TAA stop codon. The plasmidpMSATNco1 was digested with NcoI/BamH1 as recommended by themanufacturer (Roche) and the 658 bp fragment containing the perhydrolasegene was purified using standard procedures known in the art (e.g.,Sambrook et al.). The fragment was ligated using standard proceduresknown in the art (e.g., Sambrook et al.) into the T7 promoter expressionplasmid, pET16b (Novagen), also digested with NcoI/BamH1. The ligationreaction was transformed by standard procedures into E. coli Top 10cells (Invitrogen) and selected on L agar containing 100 micrograms/mlcarbenicillin overnight at 37° C. Ten colonies were picked from theseveral transformants and used to inoculate 5 ml of LB containing 100micrograms/ml carbenicillin. Cultures were grown overnight at 37° C.with shaking at 200 rpm. Plasmid DNA was purified from the culturesusing the Qiagen Quikspin kit (Qiagen). The presence of the correctfragment was determined by restriction enzyme digest with NcoI/BamH1 asdirected by the manufacturer. The correct plasmid was designatedpMSATNcoI-1 (See, FIG. 13, for the map of this plasmid). In this Figure,the following elements are indicated—LacI: gene encoding the Ladrepressor protein, located at bp1455-2534, ori: plasmid origin ofreplication at bp 4471, bla: The β-lactamase gene located at bp6089-5232; T7 promoter: located at bp1068-1052; T7 terminator: locatedat bp 259-213, per: the M. smegmatis perhydrolase gene located at981-334. The sequence of this plasmid is provided below:

(SEQ ID NO: 131) ttctcatgtttgacagcttatcatcgataagctttaatgcggtagtttatcacagttaaattgctaacgcagtcaggcaccgtgtatgaaatctaacaatgcgctcatcgtcatcctcggcaccgtcaccctggatgctgtaggcataggcttggttatgccggtactgccgggcctcttgcgggatatccggatatagttcctcctttcagcaaaaaacccctcaagacccgtttagaggccccaaggggttatgctagttattgctcagcggtggcagcagccaactcagcttcctttcgggctttgttagcagccggatccgcgcgcttacagcaggctccgcacctgttccgcgagggccaccccgagatcgcgattgttggcctcggtgaagtggattccgtcgacgccgtcggtgctgatcaccgaacccgcgtcgaagaacggcaccttcatgaacgacgcgagcgcgctgtacacgcgggcgagctcagtggtcttctgctcgccgccctcgaagatcaactggaaccaggggtgcggcatgggcgccagcggtggcggcgagaccaccagcaccttgggtgccgggtacgtggtgccgacgccgcccgcgctggtgagcacctgcgtgacgagcaccgacatgcccagcgcgatgtcgagcggggtgcgccggaagtaggccttggtgtcgttggtgcccagcatgatgatcaccaggtcgagcggcaggtgcgtcgcgaggcacgacggcaggtagctcgcgccgttgagccgcggatcggtggggtcgtcgatgttggtggtgcgcgcgctcagtccctcctcgatcacctcgaagtccgctccgagctgctgggccagcacaccggtccagcgcacgtcgggggcgaaccgctcggtgggtgccccgtcttcgacggggacccagccccaggtcagggaatcaccgaaacacagaattcgcttggccatggtatatctccttcttaaagttaaacaaaattatttctagaggggaattgttatccgctcacaattcccctatagtgagtcgtattaatttcgcgggatcgagatctcgatcctctacgccggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggccgggggactgttgggcgccatctccttgcatgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataagggagagcgtcgagatcccggacaccatcgaatggcgcaaaacctttcgcggtatggcatgatagcgcccggaagagagtcaattcagggtggtgaatgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctcccatgaagacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaagacagctcatgttatatcccgccgttaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtaagttagctcactcattaggcaccgggatctcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctctgggtcattttcggcgaggaccgctttcgctggagcgcgacgatgatcggcctgtcgcttgcggtattcggaatcttgcacgccctcgctcaagccttcgtcactggtcccgccaccaaacgtttcggcgagaagcaggccattatcgccggcatggcggccgacgcgctgggctacgtcttgctggcgttcgcgacgcgaggctggatggccttccccattatgattcttctcgcttccggcggcatcgggatgcccgcgttgcaggccatgctgtccaggcaggtagatgacgaccatcagggacagcttcaaggatcgctcgcggctcttaccagcctaacttcgatcactggaccgctgatcgtcacggcgatttatgccgcctcggcgagcacatggaacgggttggcatggattgtaggcgccgccctataccttgtctgcctccccgcgttgcgtcgcggtgcatggagccgggccacctcgacctgaatggaagccggcggcacctcgctaacggattcaccactccaagaattggagccaatcaattcttgcggagaactgtgaatgcgcaaaccaacccttggcagaacatatccatcgcgtccgccatctccagcagccgcacgcggcgcatctcgggcagcgttgggtcctggccacgggtgcgcatgatcgtgctcctgtcgttgaggacccggctaggctggcggggttgccttactggttagcagaatgaatcaccgatacgcgagcgaacgtgaagcgactgctgctgcaaaacgtctgcgacctgagcaacaacatgaatggtcttcggtttccgtgtttcgtaaagtctggaaacgcggaagtcagcgccctgcaccattatgttccggatctgcatcgcaggatgctgctggctaccctgtggaacacctacatctgtattaacgaagcgctggcattgaccctgagtgatttttctctggtcccgccgcatccataccgccagttgtttaccctcacaacgttccagtaaccgggcatgttcatcatcagtaacccgtatcgtgagcatcctctctcgtttcatcggtatcattacccccatgaacagaaatcccccttacacggaggcatcagtgaccaaacaggaaaaaaccgcccttaacatggcccgctttatcagaagccagacattaacgcttctggagaaactcaacgagctggacgcggatgaacaggcagacatctgtgaatcgcttcacgaccacgctgatgagctttaccgcagctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctgcaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaacacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgt cttcaagaa

This plasmid was transformed into the E. coli strain BL21(λDE3)pLysS(Novagen), which contains the gene encoding the T7 RNA polymerase, withselection on LA containing 100 micrograms/ml carbenicillin. Cells weregrown overnight at 37° C. One transformant was selected and the strainwas designated MSATNco1.

Production of Perhydrolase in MSATNco1-1

Production of perhydrolase was done in cell culture. For example, 5 mlof LB with carbenicillin at a concentration of 100 micrograms/ml wasinoculated with a single colony of MSATNco1 and grown overnight at 37°C. with shaking at 200 rpm. This culture was used to inoculate 100 ml ofLB with carbenicillin at a concentration of 100 micrograms/ml (in a 250ml baffled flask) to an OD₆₀₀ of 0.1. The cultures were grown at 30° C.with shaking at 200 rpm until they reached an OD₆₀₀ of 0.4. Theexpression of the perhydrolase gene was then induced by the addition of100 micromolar IPTG and the incubation continued overnight. Cultureswere harvested by centrifugation (10 min at 7000 rpm, Sorvall SS34rotor), the supernatant was removed and the pellets washed in 50 mMKPO₄, pH 6.8. The cells were centrifuged again, the supernatants removedand the wet weight of the cells was determined. The cells wereresuspended in 100 mM KPO₄ in a volume that was 4× the wet weight. Theresuspended cells were frozen at −70° C. The cells were thawed and lysedin a French Pressure cell using standard procedures known in the art.The purification steps and assessment methods are provided in Example 1.FIG. 6 provides a purification table showing the enzyme activity of theperhydrolase of the present invention through various steps in thepurification process.

M. smegmatis Perhydrolase is in an Operon

In additional experiments, it was determined that the M. smegmatisperhydrolase is part of an operon. The gene (phd) is the first gene inan operon that contains at least 2 genes, including phd, that areseparated by 10 bp (GGCTGGGGGC [SEQ ID NO:7]) not including the TAA stopcodon of phd. It is also possible that there are three genes in theoperon, with the third being either 48 bp or 61 bp to the next ORF (openreading frame). The latter two candidate genes have no significanthomology to proteins in the database.

A putative promoter was identified for M. smegmatis phd operon, TTGGGC(−35) SP (18) CCAGAT by sequence analysis and comparison with known M.smegmatis promoters (See e.g., Salazar et al., Microbiol., 149:773-784[2003]). It is not intended that the present invention be limited to anyparticular promoter and/or construct design, as it is contemplated thatother promoters and construct designs will find use in the presentinvention.

The second gene in the phd operon encodes a protein (putative PBP-3)with the sequence:

(SEQ ID NO: 9) mhlrpaltwllvvglfisvvgcssspdpadrfsafaealgrkdaaaaaaqtsdpaaaeaaitamlagmgdaanysvaaepeegddagatlkytwtwgegrdfgydttataaksgddwlitwsptylhrdltpdlrfqysedselqtpvldrtgqplmtwqtygvitverahpesaaplaallapfdpattesvtaqlnsaddrvtvmklreddlgqvrdqlaqipgvtvreqgelltadrqlsspaisgldelwhdritanagwsvylvdadgapaqqltstppkdtgpvrttldlrmqllaqqavaketrpavvvaisgstggilaaaqnpaadpqgaiafsglyppgstfktittaaaldaglatpdtpvacpgeltienrtipnddnfdlgtvplssafshscntsmaalsdelppnaltdmakdfgigvdfmvpglttvtgrvpnadnaaqrvengigqgtvtvspfglavaeaslahgstilptlvdgekttadtpsvplppnitdalrammrgtvtegtatalsdipdlggktgtaefgdnthshgwfagiagdiafatlvvggdssapavaisgdflrpalagThe corresponding DNA sequence of the gene encoding the putative PBP-3:

(SEQ ID NO: 8)atgcacttacgtcccgctctgacgtggctcctggttgtcggtctgttcatatcggtcgtcggatgttcgtcgtccccggatccggccgaccggttctcggcgttcgccgaggcgctgggccgcaaggatgcggccgcggcggccgcccagaccagcgatccggcggccgcggaggcggccatcaccgcgatgctggccgggatgggcgacgccgcgaacgtctcggtggccgccgaacccgaggaaggcgacgacgcgggcgcgacgctgaagtacacgtggacctggggtgagggccgcgacttcggctacgacaccaccgcgacggcggccaaatccggtgacgactggctgatcacctggtcccccaccgtgttgcaccgcgacctcaccccggatctgcgcttccagtacagcgaggacagcgaattgcagaccccggtgctcgaccgcaccggccagccgttgatgacatggcagaccgtcggtgtcatcactgtcgaacgcgcacatccggagtcggccgcaccgctcgccgccctgctggcgcccttcgatccgaccaccaccaccgaatcggtcaccgcacaactcaattcgacgaccgatgaccgcgtgacggtgatgaagctgcgcgaggacgatctgggtcaggtgcgcgatcagctcgcgcagatccccggcgtgaccgtgcgtgagcagggtgagctgctcaccgccgaccggcagctgtcctcgcccgccatcagcggcctggacgagctgtggcacgaccggatcaccgccaacgcgggctggtcggtgtacctggtcgacgccgacggtgcacccgcacaacagctcacgtccacgccgcccaaggacaccgggcccgtgcgcaccacgctggacctgcgcatgcaactgctcgcgcagcaggccgtggccaaggagacccgcccggccgtggtggtcgcgatctccggatcgaccgggggcatcctggccgccgcacagaacccggccgccgatccgcaaggtgcgatcgcgttttcgggcctgtacccgccggggtcgacgttcaagaccatcaccacggcggcagccctcgacgcgggcctggccaccccggacacaccggtggcctgcccgggtgagctcaccatcgagaaccgcacgatccccaacgacgacaacttcgacctgggcaccgtgccgttgtcgtcggcgttctcgcactcctgcaacaccagcatggccgccctgtccgacgagctgccgcccaacgcactgaccgacatggcaaaggacttcgggatcggcgtcgacttcatggtgcccggcctgaccaccgtgaccggccgtgtccccaacgccgacaacgccgcccagcgtgtcgagaacggcatcggccagggcaccgtgaccgtcagcccgttcggcctcgccgtcgccgaggccagcctggcgcacggttcgacgatcctgccgacgctggtcgacggcgagaagaccacggccgacaccccgtcggtgccgttgccgcccaacatcaccgacgcgctgcgcgcgatgatgcgcggaacggtcaccgagggcacggccaccgcgttgagcgacatccccgacctgggcggcaagaccggcacggcggaattcggcgacaacacgcactcgcacggctggttcgcgggcatcgcgggcgacatcgcgttcgcgacgctggtggtcggcggcgactcgtcggcaccggccgtcgcgatctcaggagacttcctgcgccccgcgctcgccggctag.

A standard BLAST search against the protein database identified homologywith several penicillin binding proteins, class 3 (PBP-3). By sequencealignment and comparison to literature (e.g., Goffin and Ghysen,Microbiol. Mol. Biol. Rev., 66:702-38 [2002]) the PBP was found tocontain the required bar codes (conserved protein sequences that definea class of proteins) to place it in the SxxK superfamily of acyltransferases, with a C-terminal domain acyl transferase and anN-terminal domain of unknown function, but with homology to the Pen^(r)(i.e., penicillin resistant) protein fusions of class B-like II and III.This penicillin binding protein acyl transferase domain does not sharesignificant homology with the perhydrolase of the present invention,although it does share homology with Co-A dependent acyl transferasesknown in the art. The amino acid sequence is provided below.

(SEQ ID NO: 10) MHLRPALTWLLVVGLFISVVGCSSSPDPADRFSAFAEALGRKDAAAAAAQTSDPAAAEAAITAMLAGMGDAANVSVAAEPEEGDDAGATLKYTWTWGEGRDFGYDTTATAAKSGDDWLITWSPTVLHRDLTPDLRFQYSEDSELQTPVLDRTGQPLMTWQTVGVITVERAHPESAAPLAALLAPFDPTTTTESVTAQLNSTTDDRVTVMKLREDDLGQVRDQLAQIPGVTVREQGELLTADRQLSSPAISGLDELWHDRITANAGWSVYLVDADGAPAQQLTSTPPKDTGPVRTTLDLRMQLLAQQAVAKETRPAVVVAISGSTGGILAAAQNPAADPQGAIAFSGLYPPGSTFKTITTAAALDAGLATPDTPVACPGELTIENRTIPNDDNFDLGTVPLSSAFSHSCNTSMAALSDELPPNALTDMAKDFGIGVDFMVPGLTTVTGRVPNADNAAQRVENGIGQGTVTVSPFGLAVAEASLAHGSTILPTLVDGEKTTADTPSVPLPPNITDALRAMMRGTVTEGTATALSDIPDLGGKTGTAEFGDNTHSHGWFAGIAGDIAFATLVVGGDSSAPAVAISGDFLRPALAG

The family-identifying bar codes provided in the above review were: (19)V (20) G/A (140) PVxDRTG (142) TxDx3Q (22) TGGxLAx4 PaxDP (13) SxxK (51)SCN (131) KTG (50) marked in bold letters in the above sequence. Theletters represent the amino acid sequence defining the bar code; thenumbers in brackets are the intervening number of amino acids betweenthe particular bar codes; “x” represents any amino acid, (i.e., theamino acids are not conserved within the bar code but the number ofamino acids (e.g., x3 corresponding to 3 intervening amino acids) isconserved). Based on these results and other data, as described herein,it is clear that the perhydrolase of the present invention represents aunique enzyme class.

Example 5 Expression of the Perhydrolase in P. citrea

In this Example, methods used to express the perhydrolase in P. citreaare described. The plasmid pMSATNcoI was transformed into P. citrea byelectroporation using the method essentially as known in the art (Seee.g., Sambrook et al., supra) except that all cultures and recovery weredone at 30° C. The transformants were plated on L agar+carbenicillin(200 μg/ml) and incubated overnight at 30° C. Three transformants werepicked for analysis. Each colony was used to inoculate a 30 ml cultureof LB+carbenicillin (200 μg/ml) and grown overnight at 30° C. withshaking at 200 rpm. The cells were pelleted by centrifugation, washedone time in 50 mM phosphate buffer pH 7.2, and finally resuspended in 4×the wet cell weight of 100 mM phosphate buffer pH 8.0. The cells werelysed by treatment with lysozyme (2 μl of a 10 mg/ml solution per one mlof P. citrea culture) at 37° C. for one hour. The cell debris waspelleted at 13,000 rpm in a microfuge for 5 min. The resultingsupernatant was used for further analysis in SDS-PAGE and Western blots,as well as assays for enzyme activity.

SDS-PAGE analysis was carried out as known in the art (See e.g.,Sambrook et al., supra) on the supernatants. Detection of theperhydrolase protein by Western blot was done using an anti-perhydrolasepolyclonal anti-sera (prepared from purified perhydrolase protein byCovance). The blot was developed as per manufacturer's suggestions usingthe ECL plus kit (Amersham).

The enzymatic activity of the expressed perhydrolase was detected by thepNB (para-nitrophenylbutyrate) assay as described in Example 1, herein.The results are provided in the

TABLE 5-1 Enzymatic Activity of Perhydrolase Expressed by P. citreaConcentration Clone OD405 Rate (mg/liter) P. citreal 3.1129 0.479487.1922 pMSATNcoI Control (P. citrea) 2.6187 −9.8312 0

The SDS-PAGE and Western blot results, as well as the assay resultsindicated that the perhydrolase is expressed by P. citrea and is active.

Example 6 Expression of the Perhydrolase in Bacillus subtilis

The perhydrolase was expressed intracellularly in B. subtilis. A varietyof promoters find use in this embodiment, including but not limited topSPAC, pAprE, pAmyE, pVeg, pHpaII. In some embodiments, the construct ispresent on a replicating plasmid (e.g., pBH1), while in otherembodiments, it is integrated into the chromosome in one or more copies.Examples of sites for integration include, but are not limited to theaprE, the amyE, the veg or the pps regions. Indeed, it is contemplatedthat other sites known to those skilled in the art will find use in thepresent invention.

A. Intracellular Expression of the Perhydrolase in Bacillus subtilisfrom a Replicating Plasmid

B. subtilis expresses a lipase/esterase encoded by the gene pnbA thathydrolyzes the pNB substrate used to detect activity of theperhydrolase. To identify B. subtilis strains expressing theperhydrolase after transformation with replicating or integratingplasmids the pnbA gene (the entire coding sequence) was first deletedfrom the desired host using the loxP cassette deletion method describedin WO 03/083125, herein incorporated by reference. It is also noted thatother strains of Bacillus may contain one or more lipases/esterasescapable of hydrolyzing the pNB or other substrate used as an indicatorfor perhydrolase activity. In some embodiments, for optimal expressionand/or activity detection it is necessary to delete one or more of thelipases/esterases from the hosts. The Bacillus subtilis strain used inthis Example has the genotype Bacillus subtilis comK pnbA (pnbAloxP-spec, aprE, nprE, degUHy32, oppA, spoIIE3501 and will be referredto as “B. subtilis pnbA” (See e.g., WO 03/083125, supra).

In these experiments, a consensus Bacillus ribosome binding site (RBS)was used. It is not intended that the consensus RBS be the only sequenceused for expression, as a non-consensus RBS also finds use in thepresent invention. The RBS of pMSATNcoI (See, Example 4) was changed toa Bacillus consensus RBS from the 16S rRNA (5′-ATAAGGAGGTGATC-3′ [SEQ IDNO:132]) of B. subtilis and a HindIII site was added to the 5′ end ofthe RBS by PCR using a primer (502rbsforward primer) containing thedesired changes. The reaction was carried out using an MJ Research PCRmachine with 30 cycles of (1 min at 95° C., 1 min at 55° C., and 1 minat 72° C.). Template DNA (pMSATrbs) was added to a 50 μl reaction (10ng) and 10 picomoles of each primer were used.

The PCR-generated phd cassette was cloned into the PCR cloning vector,pCR-Script CM (Stratagene) and transformed into E. coli Top10 cells(Invitrogen) to make pAH502R. The complete sequence of this plasmid isprovided below.

(SEQ ID NO: 133)ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattgggtaccgggccccccctcgaggtcgacggtatcgataagcttgatatcgaattcctgcagcccgggggatccgcccaagcttaaggaggtgatctagaattccatggccaagcgaattctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcacccaccgagcggttcgcccccgacgtgcgctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggagggactgagcgcgcgcaccaccaacatcgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgcgacgcacctgccgctcgacctggtgatcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgacatcgcgctgggcatgtcggtgctcgtcacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggcacccaaggtgctggtggtctcgccgccaccgctggcgcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccactgagctcgcccgcgtgtacagcgcgctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccgacggcgtcgacggaatccacttcaccgaggccaacaatcgcgatctcggggtggccctcgcggaacaggtgcggagcctgctgtaaaaggatccccgggaagcttgcatgggctagagcggccgccaccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttcgaccgaataaatacctgtgacggaagatcacttcgcagaataaataaatcctggtgtccctgttgataccgggaagccctgggccaacttttggcgaaaatgagacgttgatcggcacgtaagaggttccaactttcaccataatgaaataagatcactaccgggcgtattttttgagttgtcgagattttcaggagctaaggaagctaaaatggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattacgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatcatgccgtttgtgatggcttccatgtcggcagaatgcttaatgaattacaacagtactgcgatgagtggcagggcggggcgtaatttttttaaggcagttattggtgcccttaaacgcctggttgctacgcctgaataagtgataataagcggatgaatggcagaaattcgaaagcaaattcgacccggtcgtcggttcagggcagggtcgttaaatagccgcttatgtctattgctggtttaccggtttattgactaccggaagcagtgtgaccgtgtgcttctcaaatgcctgaggccagtttgctcaggctctccccgtggaggtaataattgacgatatgatcctttttttctgatcaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcaagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccac

Transformants were selected on L agar containing 100 μg/mlcarbenicillin. The construct was confirmed by sequencing and biochemicalassays (e.g., pNB activity assay)

Primer set for pAH502R construction:

502rbsForward primer: (SEQ ID NO: 134)5′-ccaagcttaaggaggtgatctagaattccatggccaagcgaattctg tgtttcg-3′502Reverse Primer: (SEQ ID NO: 135) 5′-ggggatccttttacagcaggctccgcacct-3′

The HindIII-RBS-phd-BamH I DNA fragment from pAH502R was cloned into thepSPAC containing vector, pMUTIN4 (See, Vagner et al., Microbiol., 144,3097-3104 [1998]) creating the construct pAH503. The complete sequenceof pAH503 is provided below:

(SEQ ID NO: 136)ataattctacacagcccagtccagactattcggcactgaaattatgggtgaagtggtcaagacctcactaggcaccttaaaaatagcgcaccctgaagaagatttatttgaggtagcccttgcctacctagcttccaagaaagatatcctaacagcacaagagcggaaagatgttttgttctacatccagaacaacctctgctaaaattcctgaaaaattttgcaaaaagttgttgactttatctacaaggtgtggcataatgtgtggaattgtgagcgctcacaattaagcttaaggaggtgatctagaattccatggccaagcgaattctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcacccaccgagcggttcgcccccgacgtgcgctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggagggactgagcgcgcgcaccaccaacatcgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgcgacgcacctgccgctcgacctggtgatcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgacatcgcgctgggcatgtcggtgctcgtcacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggctcccaaggtgctggtggtctcgccgccaccgctggcgcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccactgagctcgcccgcgtgtacagcgcgctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccgacggcgtcgacggaatccacttcaccgaggccaacaatcgcgatctcggggtggccctcgcggaacaggtgcggagcctgctgtaaaaggatccccagcttgttgatacactaatgcttttatatagggaaaaggtggtgaactactgtggaagttactgacgtaagattacgggtcgaccgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcctggtttccggcaccagaagcggtgccggaaagctggctggagtgcgatcttcctgaggccgatactgtcgtcgtcccctcaaactggcagatgcacggttacgatgcgcccatctacaccaacgtaacctatcccattacggtcaatccgccgtttgttcccacggagaatccgacgggttgttactcgctcacatttaatgttgatgaaagctggctacaggaaggccagacgcgaattatttttgatggcgttaactcggcgtttcatctgtggtgcaacgggcgctgggtcggttacggccaggacagtcgtttgccgtctgaatttgacctgagcgcatttttacgcgccggagaaaaccgcctcgcggtgatggtgctgcgttggagtgacggcagttatctggaagatcaggatatgtggcggatgagcggcattttccgtgacgtctcgttgctgcataaaccgactacacaaatcagcgatttccatgttgccactcgctttaatgatgatttcagccgcgctgtactggaggctgaagttcagatgtgcggcgagttgcgtgactacctacgggtaacagtttctttatggcagggtgaaacgcaggtcgccagcggcaccgcgcctttcggcggtgaaattatcgatgagcgtggtggttatgccgatcgcgtcacactacgtctgaacgtcgaaaacccgaaactgtggagcgccgaaatcccgaatctctatcgtgcggtggttgaactgcacaccgccgacggcacgctgattgaagcagaagcctgcgatgtcggtttccgcgaggtgcggattgaaaatggtctgctgctgctgaacggcaagccgttgctgattcgaggcgttaaccgtcacgagcatcatcctctgcatggtcaggtcatggatgagcagacgatggtgcaggatatcctgctgatgaagcagaacaactttaacgccgtgcgctgttcgcattatccgaaccatccgctgtggtacacgctgtgcgaccgctacggcctgtatgtggtggatgaagccaatattgaaacccacggcatggtgccaatgaatcgtctgaccgatgatccgcgctggctaccggcgatgagcgaacgcgtaacgcgaatggtgcagcgcgatcgtaatcacccgagtgtgatcatctggtcgctggggaatgaatcaggccacggcgctaatcacgacgcgctgtatcgctggatcaaatctgtcgatccttcccgcccggtgcagtatgaaggcggcggagccgacaccacggccaccgatattatttgcccgatgtacgcgcgcgtggatgaagaccagcccttcccggctgtgccgaaatggtccatcaaaaaatggctttcgctacctggagagacgcgcccgctgatcctttgcgaatacgcccacgcgatgggtaacagtcttggcggtttcgctaaatactggcaggcgtttcgtcagtatccccgtttacagggcggcttcgtctgggactgggtggatcagtcgctgattaaatatgatgaaaacggcaacccgtggtcggcttacggcggtgattttggcgatacgccgaacgatcgccagttctgtatgaacggtctggtctttgccgaccgcacgccgcatccagcgctgacggaagcaaaacaccagcagcagtttttccagttccgtttatccgggcaaaccatcgaagtgaccagcgaatacctgttccgtcatagcgataacgagctcctgcactggatggtggcgctggatggtaagccgctggcaagcggtgaagtgcctctggatgtcgctccacaaggtaaacagttgattgaactgcctgaactaccgcagccggagagcgccgggcaactctggctcacagtacgcgtagtgcaaccgaacgcgaccgcatggtcagaagccgggcacatcagcgcctggcagcagtggcgtctggcggaaaacctcagtgtgacgctccccgccgcgtcccacgccatcccgcatctgaccaccagcgaaatggatttttgcatcgagctgggtaataagcgttggcaatttaaccgccagtcaggctttctttcacagatgtggattggcgataaaaaacaactgctgacgccgctgcgcgatcagttcacccgtgcaccgctggataacgacattggcgtaagtgaagcgacccgcattgaccctaacgcctgggtcgaacgctggaaggcggcgggccattaccaggccgaagcagcgttgttgcagtgcacggcagatacacttgctgatgcggtgctgattacgaccgctcacgcgtggcagcatcaggggaaaaccttatttatcagccggaaaacctaccggattgatggtagtggtcaaatggcgattaccgttgatgttgaagtggcgagcgatacaccgcatccggcgcggattggcctgaactgccagctggcgcaggtagcagagcgggtaaactggctcggattagggccgcaagaaaactatcccgaccgccttactgccgcctgttttgaccgctgggatctgccattgtcagacatgtataccccgtacgtcttcccgagcgaaaacggtctgcgctgcgggacgcgcgaattgaattatggcccacaccagtggcgcggcgacttccagttcaacatcagccgctacagtcaacagcaactgatggaaaccagccatcgccatctgctgcacgcggaagaaggcacatggctgaatatcgacggtttccatatggggattggtggcgacgactcctggagcccgtcagtatcggcggaattacagctgagcgccggtcgctaccattaccagttggtctggtgtcaaaaataataataaccgggcaggccatgtctgcccgtatttcgcgtaaggaaatccattatgtactatttcaagctaattccggtggaaacgaggtcatcatttccttccgaaaaaacggttgcatttaaatcttacatatgtaatactttcaaagactacatttgtaagatttgatgtttgagtcggctgaaagatcgtacgtaccaattattgtttcgtgattgttcaagccataacactgtagggatagtggaaagagtgcttcatctggttacgatcaatcaaatattcaaacggagggagacgattttgatgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctcccatgaagacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaagacagctcatgttatatcccgccgtcaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttaggcatcgcatcctgtctcgcgtcgtcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctgcaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaacacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtcttcaagaattgatcctctagcacaaaagaaaaacgaaatgatacaccaatcagtgcaaaaaaagatataatgggagataagacggttcgtgttcgtgctgacttgcaccatatcataaaaatcgaaacagcaaagaatggcggaaacgtaaaagaagttatggaaataagacttagaagcaaacttaagagtgtgttgatagtgcagtatcttaaaattttgtataataggaattgaagttaaattagatgctaaaaatttgtaattaagaaggagtgattacatgaacaaaaatataaaatattctcaaaactttttaacgagtgaaaaagtactcaaccaaataataaaacaattgaatttaaaagaaaccgataccgtttacgaaattggaacaggtaaagggcatttaacgacgaaactggctaaaataagtaaacaggtaacgtctattgaattagacagtcatctattcaacttatcgtcagaaaaattaaaactgaatactcgtgtcactttaattcaccaagatattctacagtttcaattccctaacaaacagaggtataaaattgttgggagtattccttaccatttaagcacacaaattattaaaaaagtggtttttgaaagccatgcgtctgacatctatctgattgttgaagaaggattctacaagcgtaccttggatattcaccgaacactagggttgctcttgcacactcaagtctcgattcagcaattgcttaagctgccagcggaatgctttcatcctaaaccaaaagtaaacagtgtcttaataaaacttacccgccataccacagatgttccagataaatattggaagctatatacgtactttgtttcaaaatgggtcaatcgagaatatcgtcaactgtttactaaaaatcagtttcatcaagcaatgaaacacgccaaagtaaacaatttaagtaccgttacttatgagcaagtattgtctatttttaatagttatctattatttaacgggaggaaataattctatgagtcgcttttgtaaatttggaaagttacacgttactaaagggaatgtagataaattattaggtatactactgacagcttccaaggagctaaagaggtccctagactctagacccggggatctctgcagtcggatctggtaatgactctctagcttgaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgctctagctaagcagaaggccatcctgacggatggcctttttgcgtttctacaaactcttgttaactctagagctgcctgccgcgtttcggtgatgaagatcttcccgatgattaattaattcagaacgctcggttgccgccgggcgttttttatgcagcaatggcaagaacgttgctctaga

The construction of pAH503 was confirmed by RFLP and pNB activityassays. The pSPAC-RBS-phd DNA cassette was isolated as a BglII/SmaIdigest and then subcloned into the replicating plasmid pBH1, digestedwith BamH1/EcoRV (See e.g., EP 0275509) to create pAH505 (See, FIG. 14).The complete sequence of the plasmid is provided below.

(SEQ ID NO: 137)gatcttccaagatatcctaacagcacaagagcggaaagatgttttgttctacatccagaacaacctctgctaaaattcctgaaaaattttgcaaaaagttgttgactttatctacaaggtgtggcataatgtgtggaattgtgagcgctcacaattaagcttaaggaggtgatctagaattccatggccaagcgaattctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcacccaccgagcggttcgcccccgacgtgcgctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggagggactgagcgcgcgcaccaccaacatcgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgcgacgcacctgccgctcgacctggtgatcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgacatcgcgctgggcatgtcggtgctcgtcacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggctcccaaggtgctggtggtctcgccgccaccgctggcgcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccactgagctcgcccgcgtgtacagcgcgctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccgacggcgtcgacggaatccacttcaccgaggccaacaatcgcgatctcggggtggccctcgcggaacaggtgcggagcctgctgtaaaaggatcccatcgcatgcggtacctctagaagaagcttggagacaaggtaaaggataaaacagcacaattccaagaaaaacacgatttagaacctaaaaagaacgaatttgaactaactcataaccgagaggtaaaaaaagaacgaagtcgagatcagggaatgagtttataaaataaaaaaagcacctgaaaaggtgtctttttttgatggttttgaacttgttctttcttatcttgatacatatagaaataacgtcatttttattttagttgctgaaaggtgcgttgaagtgttggtatgtatgtgttttaaagtattgaaaacccttaaaattggttgcacagaaaaaccccatctgttaaagttataagtgactaaacaaataactaaatagatgggggtttcttttaatattatgtgtcctaatagtagcatttattcagatgaaaaatcaagggttttagtggacaagacaaaaagtggaaaagtgagaccatggagagaaaagaaaatcgctaatgttgattactttgaacttctgcatattcttgaatttaaaaaggctgaaagagtaaaagattgtgctgaaatattagagtataaacaaaatcgtgaaacaggcgaaagaaagttgtatcgagtgtggttttgtaaatccaggctttgtccaatgtgcaactggaggagagcaatgaaacatggcattcagtcacaaaaggttgttgctgaagttattaaacaaaagccaacagttcgttggttgtttctcacattaacagttaaaaatgtttatgatggcgaagaattaaataagagtttgtcagatatggctcaaggatttcgccgaatgatgcaatataaaaaaattaataaaaatcttgttggttttatgcgtgcaacggaagtgacaataaataataaagataattcttataatcagcacatgcatgtattggtatgtgtggaaccaacttattttaagaatacagaaaactacgtgaatcaaaaacaatggattcaattttggaaaaaggcaatgaaattagactatgatccaaatgtaaaagttcaaatgattcgaccgaaaaataaatataaatcggatatacaatcggcaattgacgaaactgcaaaatatcctgtaaaggatacggattttatgaccgatgatgaagaaaagaatttgaaacgtttgtctgatttggaggaaggtttacaccgtaaaaggttaatctcctatggtggtttgttaaaagaaatacataaaaaattaaaccttgatgacacagaagaaggcgatttgattcatacagatgatgacgaaaaagccgatgaagatggattttctattattgcaatgtggaattgggaacggaaaaattattttattaaagagtagttcaacaaacgggccagtttgttgaagattagatgctataattgttattaaaaggattgaaggatgcttaggaagacgagttattaatagctgaataagaacggtgctctccaaatattcttatttagaaaagcaaatctaaaattatctgaaaagggaatgagaatagtgaatggaccaataataatgactagagaagaaagaatgaagattgttcatgaaattaaggaacgaatattggataaatatggggatgatgttaaggctattggtgtttatggctctcttggtcgtcagactgatgggccctattcggatattgagatgatgtgtgtcatgtcaacagaggaagcagagttcagccatgaatggacaaccggtgagtggaaggtggaagtgaattttgatagcgaagagattctactagattatgcatctcaggtggaatcagattggccgcttacacatggtcaatttttctctattttgccgatttatgattcaggtggatacttagagaaagtgtatcaaactgctaaatcggtagaagcccaaacgttccacgatgcgatttgtgcccttatcgtagaagagctgtttgaatatgcaggcaaatggcgtaatattcgtgtgcaaggaccgacaacatttctaccatccttgactgtacaggtagcaatggcaggtgccatgttgattggtctgcatcatcgcatctgttatacgacgagcgcttcggtcttaactgaagcagttaagcaatcagatcttccttcaggttatgaccatctgtgccagttcgtaatgtctggtcaactttccgactctgagaaacttctggaatcgctagagaatttctggaatgggattcaggagtggacagaacgacacggatatatagtggatgtgtcaaaacgcataccattttgaacgatgacctctaataattgttaatcatgttggttacgtatttattaacttctcctagtattagtaattatcatggctgtcatggcgcattaacggaataaagggtgtgcttaaatcgggccattttgcgtaataagaaaaaggattaattatgagcgaattgaattaataataaggtaatagatttacattagaaaatgaaaggggattttatgcgtgagaatgttacagtctatcccggcattgccagtcggggatattaaaaagagtataggtttttattgcgataaactaggtttcactttggttcaccatgaagatggattcgcagttctaatgtgtaatgaggttcggattcatctatgggaggcaagtgatgaaggctggcgctctcgtagtaatgattcaccggtttgtacaggtgcggagtcgtttattgctggtactgctagttgccgcattgaagtagagggaattgatgaattatatcaacatattaagcctttgggcattttgcaccccaatacatcattaaaagatcagtggtgggatgaacgagactttgcagtaattgatcccgacaacaatttgattagcttttttcaacaaataaaaagctaaaatctattattaatctgttcagcaatcgggcgcgattgctgaataaaagatacgagagacctctcttgtatcttttttattttgagtggttttgtccgttacactagaaaaccgaaagacaataaaaattttattcttgctgagtctggctttcggtaagctagacaaaacggacaaaataaaaattggcaagggtttaaaggtggagattttttgagtgatcttctcaaaaaatactacctgtcccttgctgatttttaaacgagcacgagagcaaaacccccctttgctgaggtggcagagggcaggtttttttgtttcttttttctcgtaaaaaaaagaaaggtcttaaaggttttatggttttggtcggcactgccgacagcctcgcaggacacacactttatgaatataaagtatagtgtgttatactttacttggaagtggttgccggaaagagcgaaaatgcctcacatttgtgccacctaaaaaggagcgatttacatatgagttatgcagtttgtagaatgcaaaaagtgaaatcagggg

The ligation mixture for pAH505 was transformed into Bacillus subtilispnbA. Correct transformants were verified by RFLP and sequencing ofisolated plasmid DNA. One transformant was selected for analysis (B.subtilis pnbA/pAH505).

Expression of the perhydrolase in Bacillus was assayed using the pNBActivity Assay described herein, after growth of the desired strain inshake flask. The data showed that the perhydrolase was expressed in B.subtilis pnbA.

B. Intracellular Expression of the Perhydrolase in B. subtilis pnbA byIntegration into the Chromosome

An additional construct useful to determine expression of theperhydrolase (act) gene integrated into the chromosome of B. subtilispnbA involved use of the spoVG promoter, which was found to driveexpression of the perhydrolase gene in a non-replicating (i.e.,integrating plasmid). In some embodiments, one site of integration isthe aprE region of B. subtilis, although it is intended that integrationoccur at any suitable site. Indeed, it is not intended that the presentinvention be limited to this specific site nor this specific promoter,as various other suitable sites and promoters find use in the presentinvention.

The configuration of the promoter/gene at the aprE locus in thechromosome of Bacillus subtilis was as follows:

-   -   pAprE-aprE first 7 codons-translation        stop-pSpoVG-ATG-perhydrolase gene from second codon        The clone was constructed as described below. The primers used        were:

Up5′F (SEQ ID NO: 138) caggctgcgcaactgttgggaag FuaprEAct34R(SEQ ID NO: 139) agtagttcaccaccttttccctatataaaagcattagtgtatcaatttcagatccacaattttttgcttctcactctttac FuaprEAct4F (SEQ ID NO: 140)Aattgatacactaatgcttttatatagggaaaaggtggtgaactactatggccaagcgaattctgtgtttcggtg BsmI-DnAct504R (SEQ ID NO: 141)gtgagaggcattcggatccttttacagcaggctccg

PCR fusion is a technique well known in the art, in which two or morefragments of DNA are generated either by restriction digest or by PCRamplification. The fragments have overlapping segments, usually at least18 bases long. In the instance that two fragments are used, the 3′ endof fragment #1 has an overlapping sequence with the 5′ end of fragment#2. The two fragments are used as template in a PCR reaction in whichthe primer set used hybridizes to the 5′ end of fragment #1 (forwardprimer) and the 3′ end of fragment #2 (reverse primer). During theamplification, the two regions of overlap hybridize forming a singletemplate from which the two primers can amplify a full length fragment,a “fusion” of fragments #1 and #2. Multiple fragments of any length canbe used in such a reaction, limited only by the ability of the chosenpolymerase to amplify long DNA pieces.

In the current example, the above construct was made by PCR fusion oftwo PCR products the above construct was made by PCR fusion of two PCRproducts. The first was a construct with the spoVG promoter addedupstream of the phd gene. The second was the aprE promoter and first 7codons of aprE, followed by a stop codon. Regions of 20 bp overlap wereadded on the 5′ and 3′ ends of the products respectively, to allow thePCR fusion reaction. The primer set FuaprEAct4F/BsmI-DnAct504R was usedto amplify the perhydrolase gene from pAH505 as described above, whichadded the spoVG promoter sequence (contained within the primer) to the5′ end of the gene and changed the start codon from ATG to GTG. Tocreate the second product (pAprE plus the first 7 codons of aprE) forthe fusion, the primer set Up5′F/FuaprEAct34R was used to amplify afragment from pBSFNASally. FIG. 15 provides a map of this plasmid. Thecomplete sequence of pBSFNASally is provided below.

(SEQ ID NO: 142)ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattggagctccaccgcggtggcggccgctctagaactagtggatcccccgggctgcaggaattctccattttcttctgctatcaaaataacagactcgtgattttccaaacgagctttcaaaaaagcctctgccccttgcaaatcggatgcctgtctataaaattcccgatattggttaaacagcggcgcaatggcggccgcatctgatgtctttgcttggcgaatgttcatcttatttcttcctccctctcaataattttttcattctatcccttttctgtaaagtttatttttcagaatacttttatcatcatgctttgaaaaaatatcacgataatatccattgttctcacggaagcacacgcaggtcatttgaacgaattttttcgacaggaatttgccgggactcaggagcatttaacctaaaaaagcatgacatttcagcataatgaacatttactcatgtctattttcgttcttttctgtatgaaaatagttatttcgagtctctacggaaatagcgagagatgatatacctaaatagagataaaatcatctcaaaaaaatgggtctactaaaatattattccatctattacaataaattcacagaatagtcttttaagtaagtctactctgaatttttttaaaaggagagggtaaagagtgagaagcaaaaaattgtggatcagtttgctgtttgctttagcgttaatctttacgatggcgttcggcagcacatcctctgcccaggcggcagggaaatcaaacggggaaaagaaatatattgtcgggtttaaacagacaatgagcacgatgagcgccgctaagaagaaagatgtcatttctgaaaaaggcgggaaagtgcaaaagcaattcaaatatgtagacgcagcttcagctacattaaacgaaaaagctgtaaaagaattgaaaaaagacccgagcgtcgcttacgttgaagaagatcacgtagcacatgcgtacgcgcagtccgtgccttacggcgtatcacaaattaaagcccctgctctgcactctcaaggctacactggatcaaatgttaaagtagcggttatcgacagcggtatcgattcttctcatcctgatttaaaggtagcaggcggagccagcatggttccttctgaaacaaatcctttccaagacaacaactctcacggaactcacgttgccggcacagttgcggctcttaataactcaatcggtgtattaggcgttgcgccaagcgcatcactttacgctgtaaaagttctcggtgctgacggttccggccaatacagctggatcattaacggaatcgagtgggcgatcgcaaacaatatggacgttattaacatgagcctcggcggaccttctggttctgctgctttaaaagcggcagttgataaagccgttgcatccggcgtcgtagtcgttgcggcagccggtaacgaaggcacttccggcagctcaagcacagtgggctaccctggtaaatacccttctgtcattgcagtaggcgctgttgacagcagcaaccaaagagcatctttctcaagcgtaggacctgagcttgatgtcatggcacctggcgtatctatccaaagcacgcttcctggaaacaaatacggcgcgttgaacggtacatcaatggcatctccgcacgttgccggagcggctgctttgattctttctaagcacccgaactggacaaacactcaagtccgcagcagtttagaaaacaccactacaaaacttggtgattctttctactatggaaaagggctgatcaacgtacaggcggcagctcagtaaaacataaaaaaccggccttggccccgccggttttttattatttttcttcctccgcatgttcaatccgctccataatcgacggatggctccctctgaaaattttaacgagaaacggcgggttgacccggctcagtcccgtaacggccaagtcctgaaacgtctcaatcgccgcttcccggtttccggtcagctcaatgccgtaacggtcggcggcgttttcctgataccgggagacggcattcgtaatcggatcctctagagtcgatttttacaagaattagctttatataatttctgtttttctaaagttttatcagctacaaaagacagaaatgtattgcaatcttcaactaaatccatttgattctctccaatatgacgtttaataaatttctgaaatacttgatttctttgttttttctcagtatacttttccatgttataacacataaaaacaacttagttttcacaaactatgacaataaaaaaagttgctttttcccctttctatgtatgttttttactagtcatttaaaacgatacattaataggtacgaaaaagcaactttttttgcgcttaaaaccagtcataccaataacttaagggtaactagcctcgccggcaatagttacccttattatcaagataagaaagaaaaggatttttcgctacgctcaaatcctttaaaaaaacacaaaagaccacattttttaatgtggtctttattcttcaactaaagcacccattagttcaacaaacgaaaattggataaagtgggatatttttaaaatatatatttatgttacagtaatattgacttttaaaaaaggattgattctaatgaagaaagcagacaagtaagcctcctaaattcactttagataaaaatttaggaggcatatcaaatgaactttaataaaattgatttagacaattggaagagaaaagagatatttaatcattatttgaaccaacaaacgacttttagtataaccacagaaattgatattagtgttttataccgaaacataaaacaagaaggatataaattttaccctgcatttattttcttagtgacaagggtgataaactcaaatacagcttttagaactggttacaatagcgacggagagttaggttattgggataagttagagccactttatacaatttttgatggtgtatctaaaacattctctggtatttggactcctgtaaagaatgacttcaaagagttttatgatttatacctttctgatgtagagaaatataatggttcggggaaattgtttcccaaaacacctatacctgaaaatgctttttctctttctattattccatggacttcatttactgggtttaacttaaatatcaataataatagtaattaccttctacccattattacagcaggaaaattcattaataaaggtaattcaatatatttaccgctatctttacaggtacatcattctgtttgtgatggttatcatgcaggattgtttatgaactctattcaggaattgtcagataggcctaatgactggcttttataatatgagataatgccgactgtactttttacagtcggttttctaatgtcactaacctgccccgttagttgaagaaggtttttatattacagctccagatccatatccttctttttctgaaccgacttctcctttttcgcttctttattccaattgctttattgacgttgagcctcggaacccttaacaatcccaaaacttgtcgaatggtcggcttaatagctcacgctatgccgacattcgtctgcaagtttagttaagggttcttctcaacgcacaataaattttctcggcataaatgcgtggtctaatttttatttttaataaccttgatagcaaaaaatgccattccaatacaaaaccacatacctataatcgaccggaattaattctccattttcttctgctatcaaaataacagactcgtgattttccaaacgagctttcaaaaaagcctctgccccttgcaaatcggatgcctgtctataaaattcccgatattggttaaacagcggcgcaatggcggccgcatctgatgtctttgcttggcgaatgttcatcttatttcttcctccctctcaataattttttcattctatcccttttctgtaaagtttatttttcagaatacttttatcatcatgctttgaaaaaatatcacgataatatccattgttctcacggaagcacacgcaggtcatttgaacgaattttttcgacaggaatttgccgggactcaggagcatttaacctaaaaaagcatgacatttcagcataatgaacatttactcatgtctattttcgttcttttctgtatgaaaatagttatttcgagtctctacggaaatagcgagagatgatatacctaaatagagataaaatcatctcaaaaaaatgggtctactaaaatattattccatctattacaataaattcacagaatagtcttttaagtaagtctactctgaatttttttatcaagcttatcgataccgtcgacctcgagggggggcccggtacccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccac

The two PCR products were subjected to fusion PCR as known in the art tocreate the 1.5 kb fusion. The resulting fusion product was then clonedinto PCR2.1TOPO to produce pCP609 (See, FIG. 16) and sequence below).

(SEQ ID NO: 143)caggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattggagctccaccgcggtggcggccgctctagaactagtggatcccccgggctgcaggaattctccattttcttctgctatcaaaataacagactcgtgattttccaaacgagctttcaaaaaagcctctgccccttgcaaatcggatgcctgtctataaaattcccgatattggttaaacagcggcgcaatggcggccgcatctgatgtctttgcttggcgaatgttcatcttatttcttcctccctctcaataattttttcattctatcccttttctgtaaagtttatttttcagaatacttttatcatcatgctttgaaaaaatatcacgataatatccattgttctcacggaagcacacgcaggtcatttgaacgaattttttcgacaggaatttgccgggactcaggagcatttaacctaaaaaagcatgacatttcagcataatgaacatttactcatgtctattttcgttcttttctgtatgaaaatagttatttcgagtctctacggaaatagcgagagatgatatacctaaatagagataaaatcatctcaaaaaaatgggtctactaaaatattattccatctattacaataaattcacagaatagtcttttaagtaagtctactctgaatttttttaaaaggagagggtaaagagtgagaagcaaaaaattgtggatctgaaattgatacactaatgcttttatatagggaaaaggtggtgaactactatggccaagcgaattctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcacccaccgagcggttcgcccccgacgtgcgctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggagggactgagcgcgcgcaccaccaacatcgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgcgacgcacctgccgctcgacctggtgatcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgacatcgcgctgggcatgtcggtgctcgtcacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggctcccaaggtgctggtggtctcgccgccaccgctggcgcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccactgagctcgcccgcgtgtacagcgcgctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccgacggcgtcgacggaatccacttcaccgaggccaacaatcgcgatctcggggtggccctcgcggaacaggtgcggagcctgctgtaaaaggatccgaatgcctctcacaagggcgaattctgcagatatccatcacactggcggccgctcgagcatgcatctagagggcccaattcgccctatagtgagtcgtattacaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaattcagggcgcaagggctgctaaaggaagcggaacacgtagaaagccagtccgcagaaacggtgctgaccccggatgaatgtcagctactgggctatctggacaagggaaaacgcaagcgcaaagagaaagcaggtagcttgcagtgggcttacatggcgatagctagactgggcggttttatggacagcaagcgaaccggaattgccagctggggcgccctctggtaaggttgggaagccctgcaaagtaaactggatggctttcttgccgccaaggatctgatggcgcaggggatcaagatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatcccaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcttggtaccgagctcggatccactagtaacggccgccagtgtgctggaattcgccctt

The plasmid PCP609 was digested with BamH1/XmaI to release the fragmentcontaining the pAprE-aprE-stop-pSpoVG-phd construct and ligated intopBSFNASally digested with XmaI/Bcl1 to give the plasmid pCP649. FIG. 17provides a map of pCP649. The complete sequence of pCP649 is providedbelow.

(SEQ ID NO: 144)tagaactagtggatcccccgggctgcaggaattctccattttcttctgctatcaaaataacagactcgtgattttccaaacgagctttcaaaaaagcctctgccccttgcaaatcggatgcctgtctataaaattcccgatattggttaaacagcggcgcaatggcggccgcatctgatgtctttgcttggcgaatgttcatcttatttcttcctccctctcaataattttttcattctatcccttttctgtaaagtttatttttcagaatacttttatcatcatgctttgaaaaaatatcacgataatatccattgttctcacggaagcacacgcaggtcatttgaacgaattttttcgacaggaatttgccgggactcaggagcatttaacctaaaaaagcatgacatttcagcataatgaacatttactcatgtctattttcgttcttttctgtatgaaaatagttatttcgagtctctacggaaatagcgagagatgatatacctaaatagagataaaatcatctcaaaaaaatgggtctactaaaatattattccatctattacaataaattcacagaatagtcttttaagtaagtctactctgaatttttttaaaaggagagggtaaagagtgagaagcaaaaaattgtggatctgaaattgatacactaatgcttttatatagggaaaaggtggtgaactactatggccaagcgaattctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcacccaccgagcggttcgcccccgacgtgcgctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggagggactgagcgcgcgcaccaccaacatcgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgcgacgcacctgccgctcgacctggtgatcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgacatcgcgctgggcatgtcggtgctcgtcacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggctcccaaggtgctggtggtctcgccgccaccgctggcgcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccactgagctcgcccgcgtgtacagcgcgctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccgacggcgtcgacggaatccacttcaccgaggccaacaatcgcgatctcggggtggccctcgcggaacaggtgcggagcctgctgtaacggaatgcctctcacaaggatccaagccgaattctgcagatatccatcacactggcggccgctcgagcatgcatctagagtcgatttttacaagaattagctttatataatttctgtttttctaaagttttatcagctacaaaagacagaaatgtattgcaatcttcaactaaatccatttgattctctccaatatgacgtttaataaatttctgaaatacttgatttctttgttttttctcagtatacttttccatgttataacacataaaaacaacttagttttcacaaactatgacaataaaaaaagttgctttttcccctttctatgtatgttttttactagtcatttaaaacgatacattaataggtacgaaaaagcaactttttttgcgcttaaaaccagtcataccaataacttaagggtaactagcctcgccggcaatagttacccttattatcaagataagaaagaaaaggatttttcgctacgctcaaatcctttaaaaaaacacaaaagaccacattttttaatgtggtctttattcttcaactaaagcacccattagttcaacaaacgaaaattggataaagtgggatatttttaaaatatatatttatgttacagtaatattgacttttaaaaaaggattgattctaatgaagaaagcagacaagtaagcctcctaaattcactttagataaaaatttaggaggcatatcaaatgaactttaataaaattgatttagacaattggaagagaaaagagatatttaatcattatttgaaccaacaaacgacttttagtataaccacagaaattgatattagtgttttataccgaaacataaaacaagaaggatataaattttaccctgcatttattttcttagtgacaagggtgataaactcaaatacagcttttagaactggttacaatagcgacggagagttaggttattgggataagttagagccactttatacaatttttgatggtgtatctaaaacattctctggtatttggactcctgtaaagaatgacttcaaagagttttatgatttatacctttctgatgtagagaaatataatggttcggggaaattgtttcccaaaacacctatacctgaaaatgctttttctctttctattattccatggacttcatttactgggtttaacttaaatatcaataataatagtaattaccttctacccattattacagcaggaaaattcattaataaaggtaattcaatatatttaccgctatctttacaggtacatcattctgtttgtgatggttatcatgcaggattgtttatgaactctattcaggaattgtcagataggcctaatgactggcttttataatatgagataatgccgactgtactttttacagtcggttttctaatgtcactaacctgccccgttagttgaagaaggtttttatattacagctccagatccatatccttctttttctgaaccgacttctcctttttcgcttctttattccaattgctttattgacgttgagcctcggaacccttaacaatcccaaaacttgtcgaatggtcggcttaatagctcacgctatgccgacattcgtctgcaagtttagttaagggttcttctcaacgcacaataaattttctcggcataaatgcgtggtctaatttttatttttaataaccttgatagcaaaaaatgccattccaatacaaaaccacatacctataatcgacctgcaggaattaattcctccattttcttctgctatcaaaataacagactcgtgattttccaaacgagctttcaaaaaagcctctgccccttgcaaatcggatgcctgtctataaaattcccgatattggcttaaacagcggcgcaatggcggccgcatctgatgtctttgcttggcgaatgttcatcttatttcttcctccctctcaataattttttcattctatcccttttctgtaaagtttatttttcagaatacttttatcatcatgctttgaaaaaatatcacgataatatccattgttctcacggaagcacacgcaggtcatttgaacgaattttttcgacaggaatttgccgggactcaggagcatttaacctaaaaaagcatgacatttcagcataatgaacatttactcatgtctattttcgttcttttctgtatgaaaatagttatttcgagtctctacggaaatagcgagagatgatatacctaaatagagataaaatcatctcaaaaaaatgggtctactaaaatattattccatctattacaataaattcacagaatagtcttttaagtaagtctactctgaatttttttatcaagcttatcgataccgtcgacctcgagggggggcccggtacccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattggagctccaccgcggtggcggccgctc

All constructs were confirmed by sequence analysis. PCR reactions weredone using Hercules polymerase (Roche) as per the manufacturer'sdirections.

pCP649 was transformed into B. subtilis comK pnbA and integrantsselected on L agar containing chloramphenicol (5 μg/ml). The activity ofthe expressed perhydrolase was determined by the pNB activity assay asdescribed herein. The results indicated that the perhydrolase wasexpressed and active

Example 7 Expression of the Perhydrolase in Streptomyces

In this Example, experiments conducted to assess the expression of theperhydrolase in Streptomyces are described. To test expression of theperhydrolase in Streptomyces, a replicating plasmid was constructed withthe phd gene being expressed from either the glucose isomerase (GIT) orthe A4 promoter. However, it is not intended that the present inventionbe limited to these specific promoters, as any suitable promoter willfind use with the present invention. Also, although the strain used forperhydrolase expression in this Example was Streptomyces lividans TK-23,it is contemplated that any Streptomyces will find use in the presentinvention.

The Streptomyces strains were transformed and manipulated using methodsknown in the art (See e.g., Kieser et al., Practical StreptomycesGenetics, John Innes [2000]).

Construction of pSECGT-MSAT and pSECA4-MSAT

Using standard methods known in the art, the phd coding sequence (See,Example 4) was cloned into pSECGT to place the gene under control of theGI promoter. Similarly, the gene was cloned in the same plasmid with theA4 promoter using methods known in the art. Transformants were firstselected in E. coli, verified by sequence analysis, and then transformedinto S. lividans TK-23 using methods known in the art (See e.g., Kieseret al., [2000], supra). The correct clones expressed from the GIpromoter and the A4 promoter were designated “pSECGT-MSAT” and“pSECA4-phd.” The sequence of pSECGT-MSAT is provided below, while FIG.18 provides a map of the plasmid.

(SEQ ID NO: 145)ctagagtcgaccacgcaggccgccaggtagtcgacgttgatctcgcagccgagcccggccggaccggcggcgctgagcgcgaggccgacggcgggacggccggcaccggtacgcggtggcgggtcgagttcggtgagcagcccaccggcgatcaggtcgtcgacgagcgcggagacggtggcccgggtgagcccggtgacggcggcaactcccgcgcgggagagccgatctgtgctgtttgccacggtatgcagcaccagcgcgagattatgggctcgcacgctcgactgtcggacgggggcactggaacgagaagtcaggcgagccgtcacgcccttgacaatgccacatcctgagcaaataattcaaccactaaacaaatcaaccgcgtttcccggaggtaaccatggccaagcgaattctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcacccaccgagcggttcgcccccgacgtgcgctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggagggactgagcgcgcgcaccaccaacatcgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgcgacgcacctgccgctcgacctggtgatcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgacatcgcgctgggcatgtcggtgctcgtcacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggcacccaaggtgctggtggtctcgccgccaccgctggcgcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccactgagctcgcccgcgtgtacagcgcgctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccgacggcgtcgacggaatccacttcaccgaggccaacaatcgcgatctcggggtggccctcgcggaacaggtgcggagcctgctgtaacgggatccgcgagcggatcggctgaccggagcggggaggaggacgggcggccggcggaaaagtccgccggtccgctgaatcgctccccgggcacggacgtggcagtatcagcgccatgtccggcatatcccagccctccgcatgccccgaattcggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctcttgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtaagcttgcatgcctgcaggagtggggaggcacgatggccgctttggtcgacctcaacgagacgatgaagccgtggaacgacaccaccccggcggccctgctggaccacacccggcactacaccttcgacgtctgatcatcactgacgaatcgaggtcgaggaaccgagcgtccgaggaacacaggcgcttatcggttggccgcgagattcctgtcgatcctctcgtgcagcgcgattccgagggaaacggaaacgttgagagactcggtctggctcatcatggggatggaaaccgaggcggaagacgcctcctcgaacaggtcggaaggcccacccttttcgctgccgaacagcaaggccagccgatccggattgtccccgagttccttcacggaaatgtcgccatccgccttgagcgtcatcagctgcataccgctgtcccgaatgaaggcgatggcctcctcgcgaccggagagaacgacgggaagggagaagacgtaacctcggctggccctttggagacgccggtccgcgatgctggtgatgtcactgtcgaccaggatgatccccgacgctccgagcgcgagcgacgtgcgtactatcgcgccgatgttcccgacgatcttcaccccgtcgagaacgacgacgtccccacgccggctcgcgatatcgccgaacctggccgggcgagggacgcgggcgatgccgaatgtcttggccttccgctcccccttgaacaactggttgacgatcgaggagtcgatgaggcggaccggtatgttctgccgcccgcacagatccagcaactcagatggaaaaggactgctgtcgctgccgtagacctcgatgaactccaccccggccgcgatgctgtgcatgaggggctcgacgtcctcgatcaacgttgtctttatgttggatcgcgacggcttggtgacatcgatgatccgctgcaccgcgggatcggacggatttgcgatggtgtccaactcagtcatggtcgtcctaccggctgctgtgttcagtgacgcgattcctggggtgtgacaccctacgcgacgatggcggatggctgccctgaccggcaatcaccaacgcaaggggaagtcgtcgctctctggcaaagctccccgctcttccccgtccgggacccgcgcggtcgatccccgcatatgaagtattcgccttgatcagtcccggtggacgcgccagcggcccgccggagcgacggactccccgacctcgatcgtgtcgccctgagcgtccacgtagacgttgcgtgagagcaggactgggccgccgccgaccgcaccgccctcaccaccgaccgcgaccgcgccatggccgccgccgacggcctggtcgccgccgccgcccgccggttcggcgcctgacccgaccaacccccgcggggcgccggcacttcgtgctggcgccccgcccccacccaccaggagaccgaccatgaccgacttcgacggacgcctgaccgaggggaccgtgaacctggtccaggaccccaacggcggtggctggtccgcccactgcgctgagcccggttgcgactgggccgacttcgccggaccgctcggcttccagggcctcgtggccatcgctcgccgacacacgcactgaccgcacgtcaaagccccgccggatacccggcggggctctcttcggccctccaagtcacaccagccccaaggggcgtcgggagtggcggagggaacctctggcccgattggtgccaggattcccaccagaccaaagagcaacgggccggacttcgcacctccgacccgtccgctcccagactcgcgccccttagccgggcgagacaggaacgttgctcgtgcccagagtacggagcgatgccgaggcattgccagatcggcccgccgggccccgctgccactgcgggaccgcaattgcccacacaccgggcaaacggccgcgtatctactgctcagaccgctgccggatggcagcgaagcgggcgatcgcgcgtgtgacgcgagatgccgcccgaggcaaaagcgaacaccttgggaaagaaacaacagagtttcccgcacccctccgacctgcggtttctccggacggggtggatggggagagcccgagaggcgacagcctctgggaagtaggaagcacgtcgcggaccgaggctgcccgactgcggaaagccgcccggtacagccgccgccggacgctgtggcggatcagcggggacgccgcgtgcaagggctgcggccgcgccctgatggaccctgcctccggcgtgatcgtcgcccagacggcggccggaacgtccgtggtcctgggcctgatgcggtgcgggcggatctggctctgcccggtctgcgccgccacgatccggcacaagcgggccgaggagatcaccgccgccgtggtcgagtggatcaagcgcggggggaccgcctacctggtcaccttcacggcccgccatgggcacacggaccggctcgcggacctcatggacgccctccagggcacccggaagacgccggacagcccccggcggccgggcgcctaccagcgactgatcacgggcggcacgtgggccggacgccgggccaaggacgggcaccgggccgccgaccgcgagggcatccgagaccggatcgggtacgtcggcatgatccgcgcgaccgaagtcaccgtggggcagatcaacggctggcacccgcacatccacgcgatcgtcctggtcggcggccggaccgagggggagcggtccgcgaagcagatcgtcgccaccttcgagccgaccggcgccgcgctcgacgagtggcaggggcactggcggtccgtgtggaccgccgccctgcgcaaggtcaaccccgccttcacgcccgacgaccggcacggcgtcgacttcaagcggctggagaccgagcgcgacgccaacgacctcgccgagtacatcgccaagacccaggacgggaaggcgcccgccctcgaactcgcccgcgccgacctcaagacggcgaccggcgggaacgtcgccccgttcgaactcctcggacggatcggggacctgaccggcggcatgaccgaggacgacgccgccggggtcggctcgctggagtggaacctctcgcgctggcacgagtacgagcgggcaacccggggacgccgggccatcgaatggacccgctacctgcggcagatgctcgggctcgacggcggcgacaccgaggccgacgacctcgatctgctcctggcggccgacgccgacggcggggagctgcgggccggggtcgccgtgaccgaggacggatggcacgcggtcacccgccgcgccctcgacctcgaggcgacccgggccgccgaaggcaaggacggcaacgaggattcggcggccgtgggcgaacgggtgcgggaggtcctggcgctggccgacgcggccgacacagtggtggtgctcacggcgggggaggtggccgaggcgtacgccgacatgctcgccgccctcgcccagcgccgcgaggaagcaactgcacgccgacggcgagagcaggacgacgaccaggacgacgacgccgacgaccgccaggagcgggccgcccggcacatcgcccggctcgcaagtgggcccacttcgcactaactcgctcccccccgccgtacgtcatcccggtgacgtacggcgggggtcggtgacgtacgcggcgacggcggccggggtcgaagccgcgggagtaatcctgggattactcgcccggggtcggccccgccggcacttcgtgcaggcggtacctcgcgcccgactcgcctcgctacgagacgtgccgcgtacggtcgtcggccatgagcaccaccacccccagggacgccgacggcgcgaagctctgcgcctggtgcggctcggagatcaagcaatccggcgtcggccggagccgggactactgccgccgctcctgccgccagcgggcgtacgaggcccggcgccagcgcgaggcgatcgtgtccgccgtggcgtcggcagtcgctcgccgagatacgtcacgtgacgaaatgcagcagccttccattccgtcacgtgacgaaactcgggccgcaggtcagagcacggttccgcccgctccggccctgccggacccccggctgcagctcgcccggccgccggtccccctgccgtccggcccgtcccagaggcagcgtcggcggctcctgcctcccccgcccggcgccgaccgggacccgcaaaccccttgatccgctgtcgggggtgatcactacggtgggtgccgaagtgatcacggggaggactgatgcaccaccaggaccgggaccaggaccaggcgttagcggcagtgctggccgcactgctcctggtcggcgggacgctgatcgtgcgggagctcctgggcctgtggcccgccgtggcggtcggcatggcgcccgccctcgccctctacggaggcccgcccgcggcccgccggatagccgtcgcggtcgaggtccgccggttccgccggcatcttgcccaccacgatcgggcagccggatgaccggccacgacggagccgcacggctgaccagctcgacggccgccacctcatcgcggcagcaggtgctccccagcaacccacgacggggctcagggtcgcctcacgcggctcagcaccgcgacggcgggggtacggcgctccgggaggctgacaggcgctcagacggccgcgtagggccgcgagtcccccacccctccccgctgccctgtcggcgagcacaacggcgatgcccgcagtcggcggagcaggcgccacgtaaaccgcccaccgatgccgcccccgtcgtgtgcgcgggccggtcggcggccgggccggagcggggcgaagacaggagcgtcggccgggccgtgggccgggccgcgcggcccgctcgcgggccgccttgatgacgtagggaaagttgtaccgcaaaaaacgcagcctgaactagttgcgatcct

FIG. 19 provides a map of pSEGT-phdA4, while the sequence is providedbelow:

(SEQ ID NO: 146)ctagagatcgaacttcatgttcgagttcttgttcacgtagaagccggagatgtgagaggtgatctggaactgctcaccctcgttggtggtgacctggaggtaaagcaagtgacccttctggcggaggtggtaaggaacggggttccacggggagagagagatggccttgacggtcttgggaaggggagcttcngcgcgggggaggatggtcttgagagagggggagctagtaatgtcgtacttggacagggagtgctccttctccgacgcatcagccacctcagcggagatggcatcgtgcagagacagacccccggaggtaaccatggccaagcgaattctgtgtttcggtgattccctgacctggggctgggtccccgtcgaagacggggcacccaccgagcggttcgcccccgacgtgcgctggaccggtgtgctggcccagcagctcggagcggacttcgaggtgatcgaggagggactgagcgcgcgcaccaccaacatcgacgaccccaccgatccgcggctcaacggcgcgagctacctgccgtcgtgcctcgcgacgcacctgccgctcgacctggtgatcatcatgctgggcaccaacgacaccaaggcctacttccggcgcaccccgctcgacatcgcgctgggcatgtcggtgctcgtcacgcaggtgctcaccagcgcgggcggcgtcggcaccacgtacccggcacccaaggtgctggtggtctcgccgccaccgctggcgcccatgccgcacccctggttccagttgatcttcgagggcggcgagcagaagaccactgagctcgcccgcgtgtacagcgcgctcgcgtcgttcatgaaggtgccgttcttcgacgcgggttcggtgatcagcaccgacggcgtcgacggaatccacttcaccgaggccaacaatcgcgatctcggggtggccctcgcggaacaggtgcggagcctgctgtaacaatggggatccgcgagcggatcggctgaccggagcggggaggaggacgggcggccggcggaaaagtccgccggtccgctgaatcgctccccgggcacggacgtggcagtatcagcgccatgtccggcatatcccagccctccgcatgccccgaattcggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctcttgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtaagcttgcatgcctgcaggagtggggaggcacgatggccgctttggtcgacctcaacgagacgatgaagccgtggaacgacaccaccccggcggccctgctggaccacacccggcactacaccttcgacgtctgatcatcactgacgaatcgaggtcgaggaaccgagcgtccgaggaacacaggcgcttatcggttggccgcgagattcctgtcgatcctctcgtgcagcgcgattccgagggaaacggaaacgttgagagactcggtctggctcatcatggggatggaaaccgaggcggaagacgcctcctcgaacaggtcggaaggcccacccttttcgctgccgaacagcaaggccagccgatccggattgtccccgagttccttcacggaaatgtcgccatccgccttgagcgtcatcagctgcataccgctgtcccgaatgaaggcgatggcctcctcgcgaccggagagaacgacgggaagggagaagacgtaacctcggctggccctttggagacgccggtccgcgatgctggtgatgtcactgtcgaccaggatgatccccgacgctccgagcgcgagcgacgtgcgtactatcgcgccgatgttcccgacgatcttcaccccgtcgagaacgacgacgtccccacgccggctcgcgatatcgccgaacctggccgggcgagggacgcgggcgatgccgaatgtcttggccttccgctcccccttgaacaactggttgacgatcgaggagtcgatgaggcggaccggtatgttctgccgcccgcacagatccagcaactcagatggaaaaggactgctgtcgctgccgtagacctcgatgaactccaccccggccgcgatgctgtgcatgaggggctcgacgtcctcgatcaacgttgtctttatgttggatcgcgacggcttggtgacatcgatgatccgctgcaccgcgggatcggacggatttgcgatggtgtccaactcagtcatggtcgtcctaccggctgctgtgttcagtgacgcgattcctggggtgtgacaccctacgcgacgatggcggatggctgccctgaccggcaatcaccaacgcaaggggaagtcgtcgctctctggcaaagctccccgctcttccccgtccgggacccgcgcggtcgatccccgcatatgaagtattcgccttgatcagtcccggtggacgcgccagcggcccgccggagcgacggactccccgacctcgatcgtgtcgccctgagcgtccacgtagacgttgcgtgagagcaggactgggccgccgccgaccgcaccgccctcaccaccgaccgcgaccgcgccatggccgccgccgacggcctggtcgccgccgccgcccgccggttcggcgcctgacccgaccaacccccgcggggcgccggcacttcgtgctggcgccccgcccccacccaccaggagaccgaccatgaccgacttcgacggacgcctgaccgaggggaccgtgaacctggtccaggaccccaacggcggtggctggtccgcccactgcgctgagcccggttgcgactgggccgacttcgccggaccgctcggcttccagggcctcgtggccatcgctcgccgacacacgcactgaccgcacgtcaaagccccgccggatacccggcggggctctcttcggccctccaagtcacaccagccccaaggggcgtcgggagtggcggagggaacctctggcccgattggtgccaggattcccaccagaccaaagagcaacgggccggacttcgcacctccgacccgtccgctcccagactcgcgccccttagccgggcgagacaggaacgttgctcgtgcccagagtacggagcgatgccgaggcattgccagatcggcccgccgggccccgctgccactgcgggaccgcaattgcccacacaccgggcaaacggccgcgtatctactgctcagaccgctgccggatggcagcgaagcgggcgatcgcgcgtgtgacgcgagatgccgcccgaggcaaaagcgaacaccttgggaaagaaacaacagagtttcccgcacccctccgacctgcggtttctccggacggggtggatggggagagcccgagaggcgacagcctctgggaagtaggaagcacgtcgcggaccgaggctgcccgactgcggaaagccgcccggtacagccgccgccggacgctgtggcggatcagcggggacgccgcgtgcaagggctgcggccgcgccctgatggaccctgcctccggcgtgatcgtcgcccagacggcggccggaacgtccgtggtcctgggcctgatgcggtgcgggcggatctggctctgcccggtctgcgccgccacgatccggcacaagcgggccgaggagatcaccgccgccgtggtcgagtggatcaagcgcggggggaccgcctacctggtcaccttcacggcccgccatgggcacacggaccggctcgcggacctcatggacgccctccagggcacccggaagacgccggacagcccccggcggccgggcgcctaccagcgactgatcacgggcggcacgtgggccggacgccgggccaaggacgggcaccgggccgccgaccgcgagggcatccgagaccggatcgggtacgtcggcatgatccgcgcgaccgaagtcaccgtggggcagatcaacggctggcacccgcacatccacgcgatcgtcctggtcggcggccggaccgagggggagcggtccgcgaagcagatcgtcgccaccttcgagccgaccggcgccgcgctcgacgagtggcaggggcactggcggtccgtgtggaccgccgccctgcgcaaggtcaaccccgccttcacgcccgacgaccggcacggcgtcgacttcaagcggctggagaccgagcgcgacgccaacgacctcgccgagtacatcgccaagacccaggacgggaaggcgcccgccctcgaactcgcccgcgccgacctcaagacggcgaccggcgggaacgtcgccccgttcgaactcctcggacggatcggggacctgaccggcggcatgaccgaggacgacgccgccggggtcggctcgctggagtggaacctctcgcgctggcacgagtacgagcgggcaacccggggacgccgggccatcgaatggacccgctacctgcggcagatgctcgggctcgacggcggcgacaccgaggccgacgacctcgatctgctcctggcggccgacgccgacggcggggagctgcgggccggggtcgccgtgaccgaggacggatggcacgcggtcacccgccgcgccctcgacctcgaggcgacccgggccgccgaaggcaaggacggcaacgaggattcggcggccgtgggcgaacgggtgcgggaggtcctggcgctggccgacgcggccgacacagtggtggtgctcacggcgggggaggtggccgaggcgtacgccgacatgctcgccgccctcgcccagcgccgcgaggaagcaactgcacgccgacggcgagagcaggacgacgaccaggacgacgacgccgacgaccgccaggagcgggccgcccggcacatcgcccggctcgcaagtgggcccacttcgcactaactcgctcccccccgccgtacgtcatcccggtgacgtacggcgggggtcggtgacgtacgcggcgacggcggccggggtcgaagccgcgggagtaatcctgggattactcgcccggggtcggccccgccggcacttcgtgcaggcggtacctcgcgcccgactcgcctcgctacgagacgtgccgcgtacggtcgtcggccatgagcaccaccacccccagggacgccgacggcgcgaagctctgcgcctggtgcggctcggagatcaagcaatccggcgtcggccggagccgggactactgccgccgctcctgccgccagcgggcgtacgaggcccggcgccagcgcgaggcgatcgtgtccgccgtggcgtcggcagtcgctcgccgagatacgtcacgtgacgaaatgcagcagccttccattccgtcacgtgacgaaactcgggccgcaggtcagagcacggttccgcccgctccggccctgccggacccccggctgcagctcgcccggccgccggtccccctgccgtccggcccgtcccagaggcagcgtcggcggctcctgcctcccccgcccggcgccgaccgggacccgcaaaccccttgatccgctgtcgggggtgatcactacggtgggtgccgaagtgatcacggggaggactgatgcaccaccaggaccgggaccaggaccaggcgttagcggcagtgctggccgcactgctcctggtcggcgggacgctgatcgtgcgggagctcctgggcctgtggcccgccgtggcggtcggcatggcgcccgccctcgccctctacggaggcccgcccgcggcccgccggatagccgtcgcggtcgaggtccgccggttccgccggcatcttgcccaccacgatcgggcagccggatgaccggccacgacggagccgcacggctgaccagctcgacggccgccacctcatcgcggcagcaggtgctccccagcaacccacgacggggctcagggtcgcctcacgcggctcagcaccgcgacggcgggggtacggcgctccgggaggctgacaggcgctcagacggccgcgtagggccgcgagtcccccacccctccccgctgccctgtcggcgagcacaacggcgatgcccgcagtcggcggagcaggcgccacgtaaaccgcccaccgatgccgcccccgtcgtgtgcgcgggccggtcggcggccgggccggagcggggcgaagacaggagcgtcggccgggccgtgggccgggccgcgcggcccgctcgcgggccgccttgatgacgtagggaaagttgtaccgcaaaaaacgcagcctgaactagttgcgatcct

Two colonies of S. lividans TK-23 pSECA4-phd were inoculated in 10 ml ofTS medium+50 ppm thiostrepton and incubated at 37° C. with shaking at200 rpm for 2 days. Three mls of broth were used to inoculate 50 ml ofStreptomyces Production medium 1 and the culture was incubated for 4days at 37° C. with shaking at 200 rpm.

A sample was taken to assay perhydrolase activity measurement asfollows: 10 μls of 20 mg/ml lysozyme were added to 200 μl of sample.After 1 hour of incubation at 37° C., samples were centrifuged andactivity was measured using the pNB activity assay described herein.SDS-PAGE and Western blots were also prepared using both clones(pSECA4-phd and pSECGT-MSAT), as known in the art. Briefly, afterSDS-PAGE, the proteins were transferred to PVDF membrane and Westernblot analysis was conducted. The perhydrolase was detected using ananti-perhydrolase polyclonal anti-sera (1:500 dilution) prepared againstpurified perhydrolase protein by Covance. The blot was developed usingthe ECL kit from Amersham. The results indicated that Streptomyceslividans strains were capable of expressing active perhydrolase.

Example 8 Site-Scanning Mutagenesis of the M. smegmatis PerhydrolaseGene

In this Example, experiments involving site-scanning mutagenesis of theM. smegmatis perhydrolase gene are described. In these experiments, theQuikChange® site-directed mutagenesis (QC; Stratagene) kit or theQuikChange® Multi Site-Directed mutagenesis (QCMS; Stratagene) kit wasused to create site-saturation libraries at each codon in the entire M.smegmatis perhydrolase gene contained in the pMSAT-NcoI plasmid. Eachperhydrolase codon was mutagenized by replacement with the NNGC (NNS; 32combinations) degenerate codon, which encodes for all 20 amino acids andone stop codon. In the case of the QC method, complementary overlappingprimers were designed for each codon of interest with 18 bases flankingthe NNS codon (See, Tables 8-1 and 8-2). A comparison of cartridgepurified versus unpurified primers (desalted only) revealed a betterrepresentation of amino acids in the libraries made with purifiedprimers (15-19 amino acids versus 11-16 with unpurified primers). Thus,a majority of the libraries were created with the QC method and purifiedprimers. A small number of the libraries were made using the QCMS methodand a single 5′ phosphorylated forward primer containing 18 basesflanking both sides of the NNS codon (See, Table 8-1), however thismethod resulted in a greater wild type background and fewer amino acidsubstitutions per site compared to the QC methods. Libraries “nsa301”and “nsa302” were made using the QCMS method, but a trinucleotide mixmade up of a single codon for each of the 20 amino acids (i.e., ratherthan 32 possibilities encoded by NNS for the 20 amino acids) wasincorporated within the primers at the sites of interest.

TABLE 8-1 Site-Saturation Forward Primers Residue Primer Primer SequenceM1 nsa202F taacaggaggaattaaccnnsgccaagcgaattctgtgt (SEQ ID NO: 147) A2nsa203F caggaggaattaaccatgnnsaagcgaattctgtgtttc (SEQ ID NO: 148) K3nsa204F gaggaattaaccatggccnnscgaattctgtgtttcggt (SEQ ID NO: 149) R4nsa205F gaattaaccatggccaagnnsattctgtgtttcggtgat (SEQ ID NO: 150) I5nsa206F ttaaccatggccaagcgannsctgtgtttcggtgattcc (SEQ ID NO: 151) L6nsa207F accatggccaagcgaattnnstgtttcggtgattccctg (SEQ ID NO: 152) C7nsa208F atggccaagcgaattctgnnsttcggtgattccctgacc (SEQ ID NO: 153) F8nsa209F gccaagcgaattctgtgtnnsggtgattccctgacctgg (SEQ ID NO: 154) G9nsa210F aagcgaattctgtgtttcnnsgattccctgacctggggc (SEQ ID NO: 155) D10nsa168F cgaattctgtgtttcggtnnstccctgacctggggctgg (SEQ ID NO: 156) S11nsa212F attctgtgtttcggtgatnnsctgacctggggctgggtc (SEQ ID NO: 157) L12nsa169F ctgtgtttcggtgattccnnsacctggggctgggtcccc (SEQ ID NO: 158) T13nsa170F tgtttcggtgattccctgnnstggggctgggtccccgtc (SEQ ID NO: 159) W14nsa171F ttcggtgattccctgaccnnsggctgggtccccgtcgaa (SEQ ID NO: 160) G15nsa216F ggtgattccctgacctggnnstgggtccccgtcgaagac (SEQ ID NO: 161) W16nsa172F gattccctgacctggggcnnsgtccccgtcgaagacggg (SEQ ID NO: 162) V17nsa218F tccctgacctggggctggnnscccgtcgaagacggggca (SEQ ID NO: 163) P18nsa219F ctgacctggggctgggtcnnsgtcgaagacggggcaccc (SEQ ID NO: 164) V19nsa220F acctggggctgggtccccnnsgaagacggggcacccacc (SEQ ID NO: 165) E20nsa221F tggggctgggtccccgtcnnsgacggggcacccaccgag (SEQ ID NO: 166) D21nsa222F ggctgggtccccgtcgaannsggggcacccaccgagcgg (SEQ ID NO: 167) G22nsa223F tgggtccccgtcgaagacnnsgcacccaccgagcggttc (SEQ ID NO: 168) A23nsa224F gtccccgtcgaagacgggnnscccaccgagcggttcgcc (SEQ ID NO: 169) P24nsa191F cccgtcgaagacggggcannsaccgagcggttcgccccc (SEQ ID NO: 170) T25nsa192F gtcgaagacggggcacccnnsgagcggttcgcccccgac (SEQ ID NO: 171) E26nsa227F gaagacggggcacccaccnnscggttcgcccccgacgtg (SEQ ID NO: 172) R27nsa228F gacggggcacccaccgagnnsttcgcccccgacgtgcgc (SEQ ID NO: 173) F28nsa229F ggggcacccaccgagcggnnsgcccccgacgtgcgctgg (SEQ ID NO: 174) A29nsa230F gcacccaccgagcggttcnnscccgacgtgcgctggacc (SEQ ID NO: 175) P30nsa231F cccaccgagcggttcgccnnsgacgtgcgctggaccggt (SEQ ID NO: 176) D31nsa232F accgagcggttcgcccccnnsgtgcgctggaccggtgtg (SEQ ID NO: 177) V32nsa233F gagcggttcgcccccgacnnscgctggaccggtgtgctg (SEQ ID NO: 178) R33nsa234F cggttcgcccccgacgtgnnstggaccggtgtgctggcc (SEQ ID NO: 179) W34nsa235F ttcgcccccgacgtgcgcnnsaccggtgtgctggcccag (SEQ ID NO: 180) T35nsa236F gcccccgacgtgcgctggnnsggtgtgctggcccagcag (SEQ ID NO: 181) G36nsa237F cccgacgtgcgctggaccnnsgtgctggcccagcagctc (SEQ ID NO: 182) V37nsa238F gacgtgcgctggaccggtnnsctggcccagcagctcgga (SEQ ID NO: 183) L38nsa239F gtgcgctggaccggtgtgnnsgcccagcagctcggagcg (SEQ ID NO: 184) A39nsa240F cgctggaccggtgtgctgnnscagcagctcggagcggac (SEQ ID NO: 185) Q40nsa241F tggaccggtgtgctggccnnscagctcggagcggacttc (SEQ ID NO: 186) Q41nsa242F accggtgtgctggcccagnnsctcggagcggacttcgag (SEQ ID NO: 187) L42nsa243F ggtgtgctggcccagcagnnsggagcggacttcgaggtg (SEQ ID NO: 188) G43nsa244F gtgctggcccagcagctcnnsgcggacttcgaggtgatc (SEQ ID NO: 189) A44nsa245F ctggcccagcagctcggannsgacttcgaggtgatcgag (SEQ ID NO: 190) D45nsa246F gcccagcagctcggagcgnnsttcgaggtgatcgaggag (SEQ ID NO: 191) F46nsa247F cagcagctcggagcggacnnsgaggtgatcgaggaggga (SEQ ID NO: 192) E47nsa248F cagctcggagcggacttcnnsgtgatcgaggagggactg (SEQ ID NO: 193) V48nsa249F ctcggagcggacttcgagnnsatcgaggagggactgagc (SEQ ID NO: 194) I49nsa250F ggagcggacttcgaggtgnnsgaggagggactgagcgcg (SEQ ID NO: 195) E50nsa251F gcggacttcgaggtgatcnnsgagggactgagcgcgcgc (SEQ ID NO: 196) E51nsa252F gacttcgaggtgatcgagnnsggactgagcgcgcgcacc (SEQ ID NO: 197) G52nsa253F ttcgaggtgatcgaggagnnsctgagcgcgcgcaccacc (SEQ ID NO: 198) L53nsa193F gaggtgatcgaggagggannsagcgcgcgcaccaccaac (SEQ ID NO: 199) S54nsa173F gtgatcgaggagggactgnnsgcgcgcaccaccaacatc (SEQ ID NO: 200) A55nsa174F atcgaggagggactgagcnnscgcaccaccaacatcgac (SEQ ID NO: 201) R56nsa257F gaggagggactgagcgcgnnsaccaccaacatcgacgac (SEQ ID NO: 202) T57nsa258F gagggactgagcgcgcgcnnsaccaacatcgacgacccc (SEQ ID NO: 203) T58nsa259F ggactgagcgcgcgcaccnnsaacatcgacgaccccacc (SEQ ID NO: 204) N59nsa260F ctgagcgcgcgcaccaccnnsatcgacgaccccaccgat (SEQ ID NO: 205) I60nsa261F agcgcgcgcaccaccaacnnsgacgaccccaccgatccg (SEQ ID NO: 206) D61nsa262F gcgcgcaccaccaacatcnnsgaccccaccgatccgcgg (SEQ ID NO: 207) D62nsa263F cgcaccaccaacatcgacnnscccaccgatccgcggctc (SEQ ID NO: 208) P63nsa264F accaccaacatcgacgacnnsaccgatccgcggctcaac (SEQ ID NO: 209) T64nsa194F accaacatcgacgaccccnnsgatccgcggctcaacggc (SEQ ID NO: 210) D65nsa195F aacatcgacgaccccaccnnsccgcggctcaacggcgcg (SEQ ID NO: 211) P66nsa267F atcgacgaccccaccgatnnscggctcaacggcgcgagc (SEQ ID NO: 212) R67nsa196F gacgaccccaccgatccgnnsctcaacggcgcgagctac (SEQ ID NO: 213) L68nsa269F gaccccaccgatccgcggnnsaacggcgcgagctacctg (SEQ ID NO: 214) N69nsa270F cccaccgatccgcggctcnnsggcgcgagctacctgccg (SEQ ID NO: 215) G70nsa271F accgatccgcggctcaacnnsgcgagctacctgccgtcg (SEQ ID NO: 216) A71nsa272F gatccgcggctcaacggcnnsagctacctgccgtcgtgc (SEQ ID NO: 217) S72nsa273F ccgcggctcaacggcgcgnnstacctgccgtcgtgcctc (SEQ ID NO: 218) Y73nsa274F cggctcaacggcgcgagcnnsctgccgtcgtgcctcgcg (SEQ ID NO: 219) L74nsa275F ctcaacggcgcgagctacnnsccgtcgtgcctcgcgacg (SEQ ID NO: 220) P75nsa276F aacggcgcgagctacctgnnstcgtgcctcgcgacgcac (SEQ ID NO: 221) S76nsa277F ggcgcgagctacctgccgnnstgcctcgcgacgcacctg (SEQ ID NO: 222) C77nsa278F gcgagctacctgccgtcgnnsctcgcgacgcacctgccg (SEQ ID NO: 223) L78nsa279F agctacctgccgtcgtgcnnsgcgacgcacctgccgctc (SEQ ID NO: 224) A79nsa280F tacctgccgtcgtgcctcnnsacgcacctgccgctcgac (SEQ ID NO: 225) T80nsa281F ctgccgtcgtgcctcgcgnnscacctgccgctcgacctg (SEQ ID NO: 226) H81nsa282F ccgtcgtgcctcgcgacgnnsctgccgctcgacctggtg (SEQ ID NO: 227) L82nsa283F tcgtgcctcgcgacgcacnnsccgctcgacctggtgatc (SEQ ID NO: 228) P83nsa284F tgcctcgcgacgcacctgnnsctcgacctggtgatcatc (SEQ ID NO: 229) L84nsa285F ctcgcgacgcacctgccgnnsgacctggtgatcatcatg (SEQ ID NO: 230) D85nsa286F gcgacgcacctgccgctcnnsctggtgatcatcatgctg (SEQ ID NO: 231) L86nsa287F acgcacctgccgctcgacnnsgtgatcatcatgctgggc (SEQ ID NO: 232) V87nsa288F cacctgccgctcgacctgnnsatcatcatgctgggcacc (SEQ ID NO: 233) I88nsa289F ctgccgctcgacctggtgnnsatcatgctgggcaccaac (SEQ ID NO: 234) I89nsa290F ccgctcgacctggtgatcnnsatgctgggcaccaacgac (SEQ ID NO: 235) M90nsa291F ctcgacctggtgatcatcnnsctgggcaccaacgacacc (SEQ ID NO: 236) L91nsa292F gacctggtgatcatcatgnnsggcaccaacgacaccaag (SEQ ID NO: 237) G92nsa293F ctggtgatcatcatgctgnnsaccaacgacaccaaggcc (SEQ ID NO: 238) T93nsa294F gtgatcatcatgctgggcnnsaacgacaccaaggcctac (SEQ ID NO: 239) N94nsa175F atcatcatgctgggcaccnnsgacaccaaggcctacttc (SEQ ID NO: 240) D95nsa197F atcatgctgggcaccaacnnsaccaaggcctacttccgg (SEQ ID NO: 241) T96nsa297F atgctgggcaccaacgacnnsaaggcctacttccggcgc (SEQ ID NO: 242) K97nsa176F ctgggcaccaacgacaccnnsgcctacttccggcgcacc (SEQ ID NO: 243) A98nsa299F ggcaccaacgacaccaagnnstacttccggcgcaccccg (SEQ ID NO: 244) Y99nsa177F accaacgacaccaaggccnnsttccggcgcaccccgctc (SEQ ID NO: 245) F100nsa301F aacgacaccaaggcctacXXXcggcgcaccccgctcgac (SEQ ID NO: 246) R101nsa302F gacaccaaggcctacttcXXXcgcaccccgctcgacatc (SEQ ID NO: 247) R102nsa303F accaaggcctacttccggnnsaccccgctcgacatcgcg (SEQ ID NO: 248) T103nsa304F aaggcctacttccggcgcnnsccgctcgacatcgcgctg (SEQ ID NO: 249) P104nsa305F gcctacttccggcgcaccnnsctcgacatcgcgctgggc (SEQ ID NO: 250) L105nsa306F tacttccggcgcaccccgnnsgacatcgcgctgggcatg (SEQ ID NO: 251) D106nsa307F ttccggcgcaccccgctcnnsatcgcgctgggcatgtcg (SEQ ID NO: 252) I107nsa308F cggcgcaccccgctcgacnnsgcgctgggcatgtcggtg (SEQ ID NO: 253) A108nsa309F cgcaccccgctcgacatcnnsctgggcatgtcggtgctc (SEQ ID NO: 254) L109nsa310F accccgctcgacatcgcgnnsggcatgtcggtgctcgtc (SEQ ID NO: 255) G110nsa311F ccgctcgacatcgcgctgnnsatgtcggtgctcgtcacg (SEQ ID NO: 256) M111nsa312F ctcgacatcgcgctgggcnnstcggtgctcgtcacgcag (SEQ ID NO: 257) S112nsa313F gacatcgcgctgggcatgnnsgtgctcgtcacgcaggtg (SEQ ID NO: 258) V113nsa314F atcgcgctgggcatgtcgnnsctcgtcacgcaggtgctc (SEQ ID NO: 259) L114nsa315F gcgctgggcatgtcggtgnnsgtcacgcaggtgctcacc (SEQ ID NO: 260) V115nsa316F ctgggcatgtcggtgctcnnsacgcaggtgctcaccagc (SEQ ID NO: 261) T116nsa317F ggcatgtcggtgctcgtcnnscaggtgctcaccagcgcg (SEQ ID NO: 262) Q117nsa318F atgtcggtgctcgtcacgnnsgtgctcaccagcgcgggc (SEQ ID NO: 263) V118nsa319F tcggtgctcgtcacgcagnnsctcaccagcgcgggcggc (SEQ ID NO: 264) L119nsa320F gtgctcgtcacgcaggtgnnsaccagcgcgggcggcgtc (SEQ ID NO: 265) T120nsa321F ctcgtcacgcaggtgctcnnsagcgcgggcggcgtcggc (SEQ ID NO: 266) S121nsa322F gtcacgcaggtgctcaccnnsgcgggcggcgtcggcacc (SEQ ID NO: 267) A122nsa323F acgcaggtgctcaccagcnnsggcggcgtcggcaccacg (SEQ ID NO: 268) G123nsa324F caggtgctcaccagcgcgnnsggcgtcggcaccacgtac (SEQ ID NO: 269) G124nsa325F gtgctcaccagcgcgggcnnsgtcggcaccacgtacccg (SEQ ID NO: 270) V125nsa198F ctcaccagcgcgggcggcnnsggcaccacgtacccggca (SEQ ID NO: 271) G126nsa327F accagcgcgggcggcgtcnnsaccacgtacccggcaccc (SEQ ID NO: 272) T127nsa328F agcgcgggcggcgtcggcnnsacgtacccggcacccaag (SEQ ID NO: 273) T128nsa329F gcgggcggcgtcggcaccnnstacccggcacccaaggtg (SEQ ID NO: 274) Y129nsa330F ggcggcgtcggcaccacgnnsccggcacccaaggtgctg (SEQ ID NO: 275) P130nsa331F ggcgtcggcaccacgtacnnsgcacccaaggtgctggtg (SEQ ID NO: 276) A131nsa332F gtcggcaccacgtacccgnnscccaaggtgctggtggtc (SEQ ID NO: 277) P132nsa333F ggcaccacgtacccggcannsaaggtgctggtggtctcg (SEQ ID NO: 278) K133nsa334F accacgtacccggcacccnnsgtgctggtggtctcgccg (SEQ ID NO: 279) V134nsa335F acgtacccggcacccaagnnsctggtggtctcgccgcca (SEQ ID NO: 280) L135nsa336F tacccggcacccaaggtgnnsgtggtctcgccgccaccg (SEQ ID NO: 281) V136nsa337F ccggcacccaaggtgctgnnsgtctcgccgccaccgctg (SEQ ID NO: 282) V137nsa338F gcacccaaggtgctggtgnnstcgccgccaccgctggcg (SEQ ID NO: 283) S138nsa339F cccaaggtgctggtggtcnnsccgccaccgctggcgccc (SEQ ID NO: 284) P139nsa340F aaggtgctggtggtctcgnnsccaccgctggcgcccatg (SEQ ID NO: 285) P140nsa341F gtgctggtggtctcgccgnnsccgctggcgcccatgccg (SEQ ID NO: 286) P141nsa342F ctggtggtctcgccgccannsctggcgcccatgccgcac (SEQ ID NO: 287) L142nsa343F gtggtctcgccgccaccgnnsgcgcccatgccgcacccc (SEQ ID NO: 288) A143nsa344F gtctcgccgccaccgctgnnscccatgccgcacccctgg (SEQ ID NO: 289) P144nsa345F tcgccgccaccgctggcgnnsatgccgcacccctggttc (SEQ ID NO: 290) M145nsa346F ccgccaccgctggcgcccnnsccgcacccctggttccag (SEQ ID NO: 291) P146nsa178F ccaccgctggcgcccatgnnscacccctggttccagttg (SEQ ID NO: 292) H147nsa348F ccgctggcgcccatgccgnnsccctggttccagttgatc (SEQ ID NO: 293) P148nsa199F ctggcgcccatgccgcacnnstggttccagttgatcttc (SEQ ID NO: 294) W149nsa179F gcgcccatgccgcaccccnnsttccagttgatcttcgag (SEQ ID NO: 295) F150nsa180F cccatgccgcacccctggnnscagttgatcttcgagggc (SEQ ID NO: 296) Q151nsa352F atgccgcacccctggttcnnsttgatcttcgagggcggc (SEQ ID NO: 297) L152nsa353F ccgcacccctggttccagnnsatcttcgagggcggcgag (SEQ ID NO: 298) I153nsa200F cacccctggttccagttgnnsttcgagggcggcgagcag (SEQ ID NO: 299) F154nsa201F ccctggttccagttgatcnnsgagggcggcgagcagaag (SEQ ID NO: 300) E155nsa356F tggttccagttgatcttcnnsggcggcgagcagaagacc (SEQ ID NO: 301) G156nsa357F ttccagttgatcttcgagnnsggcgagcagaagaccact (SEQ ID NO: 302) G157nsa358F cagttgatcttcgagggcnnsgagcagaagaccactgag (SEQ ID NO: 303) E158nsa359F ttgatcttcgagggcggcnnscagaagaccactgagctc (SEQ ID NO: 304) Q159nsa360F atcttcgagggcggcgagnnsaagaccactgagctcgcc (SEQ ID NO: 305) K160nsa361F ttcgagggcggcgagcagnnsaccactgagctcgcccgc (SEQ ID NO: 306) T161nsa362F gagggcggcgagcagaagnnsactgagctcgcccgcgtg (SEQ ID NO: 307) T162nsa363F ggcggcgagcagaagaccnnsgagctcgcccgcgtgtac (SEQ ID NO: 308) E163nsa364F ggcgagcagaagaccactnnsctcgcccgcgtgtacagc (SEQ ID NO: 309) L164nsa365F gagcagaagaccactgagnnsgcccgcgtgtacagcgcg (SEQ ID NO: 310) A165nsa366F cagaagaccactgagctcnnscgcgtgtacagcgcgctc (SEQ ID NO: 311) R166nsa367F aagaccactgagctcgccnnsgtgtacagcgcgctcgcg (SEQ ID NO: 312) V167nsa368F accactgagctcgcccgcnnstacagcgcgctcgcgtcg (SEQ ID NO: 313) Y168nsa369F actgagctcgcccgcgtgnnsagcgcgctcgcgtcgttc (SEQ ID NO: 314) S169nsa370F gagctcgcccgcgtgtacnnsgcgctcgcgtcgttcatg (SEQ ID NO: 315) A170nsa371F ctcgcccgcgtgtacagcnnsctcgcgtcgttcatgaag (SEQ ID NO: 316) L171nsa372F gcccgcgtgtacagcgcgnnsgcgtcgttcatgaaggtg (SEQ ID NO: 317) A172nsa373F cgcgtgtacagcgcgctcnnstcgttcatgaaggtgccg (SEQ ID NO: 318) S173nsa374F gtgtacagcgcgctcgcgnnsttcatgaaggtgccgttc (SEQ ID NO: 319) F174nsa375F tacagcgcgctcgcgtcgnnsatgaaggtgccgttcttc (SEQ ID NO: 320) M175nsa376F agcgcgctcgcgtcgttcnnsaaggtgccgttcttcgac (SEQ ID NO: 321) K176nsa377F gcgctcgcgtcgttcatgnnsgtgccgttcttcgacgcg (SEQ ID NO: 322) V177nsa378F ctcgcgtcgttcatgaagnnsccgttcttcgacgcgggt (SEQ ID NO: 323) P178nsa379F gcgtcgttcatgaaggtgnnsttcttcgacgcgggttcg (SEQ ID NO: 324) F179nsa380F tcgttcatgaaggtgccgnnsttcgacgcgggttcggtg (SEQ ID NO: 325) F180nsa381F ttcatgaaggtgccgttcnnsgacgcgggttcggtgatc (SEQ ID NO: 326) D181nsa382F atgaaggtgccgttcttcnnsgcgggttcggtgatcagc (SEQ ID NO: 327) A182nsa383F aaggtgccgttcttcgacnnsggttcggtgatcagcacc (SEQ ID NO: 328) G183nsa384F gtgccgttcttcgacgcgnnstcggtgatcagcaccgac (SEQ ID NO: 329) S184nsa385F ccgttcttcgacgcgggtnnsgtgatcagcaccgacggc (SEQ ID NO: 330) V185nsa386F ttcttcgacgcgggttcgnnsatcagcaccgacggcgtc (SEQ ID NO: 331) I186nsa387F ttcgacgcgggttcggtgnnsagcaccgacggcgtcgac (SEQ ID NO: 332) S187nsa388F gacgcgggttcggtgatcnnsaccgacggcgtcgacgga (SEQ ID NO: 333) T188nsa389F gcgggttcggtgatcagcnnsgacggcgtcgacggaatc (SEQ ID NO: 334) D189nsa390F ggttcggtgatcagcaccnnsggcgtcgacggaatccac (SEQ ID NO: 335) G190nsa391F tcggtgatcagcaccgacnnsgtcgacggaatccacttc (SEQ ID NO: 336) V191nsa392F gtgatcagcaccgacggcnnsgacggaatccacttcacc (SEQ ID NO: 337) D192nsa393F atcagcaccgacggcgtcnnsggaatccacttcaccgag (SEQ ID NO: 338) G193nsa394F agcaccgacggcgtcgacnnsatccacttcaccgaggcc (SEQ ID NO: 339) I194nsa181F accgacggcgtcgacggannscacttcaccgaggccaac (SEQ ID NO: 340) H195nsa396F gacggcgtcgacggaatcnnsttcaccgaggccaacaat (SEQ ID NO: 341) F196nsa182F ggcgtcgacggaatccacnnsaccgaggccaacaatcgc (SEQ ID NO: 342) T197nsa398F gtcgacggaatccacttcnnsgaggccaacaatcgcgat (SEQ ID NO: 343) E198nsa399F gacggaatccacttcaccnnsgccaacaatcgcgatctc (SEQ ID NO: 344) A199nsa400F ggaatccacttcaccgagnnsaacaatcgcgatctcggg (SEQ ID NO: 345) N200nsa401F atccacttcaccgaggccnnsaatcgcgatctcggggtg (SEQ ID NO: 346) N201nsa402F cacttcaccgaggccaacnnscgcgatctcggggtggcc (SEQ ID NO: 347) R202nsa403F ttcaccgaggccaacaatnnsgatctcggggtggccctc (SEQ ID NO: 348) D203nsa404F accgaggccaacaatcgcnnsctcggggtggccctcgcg (SEQ ID NO: 349) L204nsa405F gaggccaacaatcgcgatnnsggggtggccctcgcggaa (SEQ ID NO: 350) G205nsa406F gccaacaatcgcgatctcnnsgtggccctcgcggaacag (SEQ ID NO: 351) V206nsa407F aacaatcgcgatctcgggnnsgccctcgcggaacaggtg (SEQ ID NO: 352) A207nsa408F aatcgcgatctcggggtgnnsctcgcggaacaggtgcag (SEQ ID NO: 353) L208nsa409F cgcgatctcggggtggccnnsgcggaacaggtgcagagc (SEQ ID NO: 354) A209nsa410F gatctcggggtggccctcnnsgaacaggtgcagagcctg (SEQ ID NO: 355) E210nsa411F ctcggggtggccctcgcgnnscaggtgcagagcctgctg (SEQ ID NO: 356) Q211nsa412F ggggtggccctcgcggaannsgtgcagagcctgctgtaa (SEQ ID NO: 357) V212nsa413F gtggccctcgcggaacagnnscagagcctgctgtaaaag (SEQ ID NO: 358) Q213nsa414F gccctcgcggaacaggtgnnsagcctgctgtaaaagggc (SEQ ID NO: 359) S214nsa415F ctcgcggaacaggtgcagnnsctgctgtaaaagggcgaa (SEQ ID NO: 360) L215nsa416F gcggaacaggtgcagagcnnsctgtaaaagggcgaattc (SEQ ID NO: 361) L216nsa417F gaacaggtgcagagcctgnnstaaaagggcgaattctgc (SEQ ID NO: 362)

TABLE 8-2 Site-Saturation Reverse Primer Sequences Residue Primer PrimerSequence M1 nsa202R ACACAGAATTCGCTTGGCSNNGGTTAATTCCTCCTGTTA (SEQ ID NO:363) A2 nsa203R GAAACACAGAATTCGCTTSNNCATGGTTAATTCCTCCTG (SEQ ID NO: 364)K3 nsa204R ACCGAAACACAGAATTCGSNNGGCCATGGTTAATTCCTC (SEQ ID NO: 365) R4nsa205R ATCACCGAAACACAGAATSNNCTTGGCCATGGTTAATTC (SEQ ID NO: 366) I5nsa206R GGAATCACCGAAACACAGSNNTCGCTTGGCCATGGTTAA (SEQ ID NO: 367) L6nsa207R CAGGGAATCACCGAAACASNNAATTCGCTTGGCCATGGT (SEQ ID NO: 368) C7nsa208R GGTCAGGGAATCACCGAASNNCAGAATTCGCTTGGCCAT (SEQ ID NO: 369) F8nsa209R CCAGGTCAGGGAATCACCSNNACACAGAATTCGCTTGGC (SEQ ID NO: 370) G9nsa210R GCCCCAGGTCAGGGAATCSNNGAAACACAGAATTCGCTT (SEQ ID NO: 371) D10nsa168R CCAGCCCCAGGTCAGGGASNNACCGAAACACAGAATTCG (SEQ ID NO: 372) S11nsa212R GACCCAGCCCCAGGTCAGSNNATCACCGAAACACAGAAT (SEQ ID NO: 373) L12nsa169R GGGGACCCAGCCCCAGGTSNNGGAATCACCGAAACACAG (SEQ ID NO: 374) T13nsa170R GACGGGGACCCAGCCCCASNNCAGGGAATCACCGAAACA (SEQ ID NO: 375) W14nsa171R TTCGACGGGGACCCAGCCSNNGGTCAGGGAATCACCGAA (SEQ ID NO: 376) G15nsa216R GTCTTCGACGGGGACCCASNNCCAGGTCAGGGAATCACC (SEQ ID NO: 377) W16nsa172R CCCGTCTTCGACGGGGACSNNGCCCCAGGTCAGGGAATC (SEQ ID NO: 378) V17nsa218R TGCCCCGTCTTCGACGGGSNNCCAGCCCCAGGTCAGGGA (SEQ ID NO: 379) P18nsa219R GGGTGCCCCGTCTTCGACSNNGACCCAGCCCCAGGTCAG (SEQ ID NO: 380) V19nsa220R GGTGGGTGCCCCGTCTTCSNNGGGGACCCAGCCCCAGGT (SEQ ID NO: 381) E20nsa221R CTCGGTGGGTGCCCCGTCSNNGACGGGGACCCAGCCCCA (SEQ ID NO: 382) D21nsa222R CCGCTCGGTGGGTGCCCCSNNTTCGACGGGGACCCAGCC (SEQ ID NO: 383) G22nsa223R GAACCGCTCGGTGGGTGCSNNGTCTTCGACGGGGACCCA (SEQ ID NO: 384) A23nsa224R GGCGAACCGCTCGGTGGGSNNCCCGTCTTCGACGGGGAC (SEQ ID NO: 385) P24nsa191R GGGGGCGAACCGCTCGGTSNNTGCCCCGTCTTCGACGGG (SEQ ID NO: 386) T25nsa192R GTCGGGGGCGAACCGCTCSNNGGGTGCCCCGTCTTCGAC (SEQ ID NO: 387) E26nsa227R CACGTCGGGGGCGAACCGSNNGGTGGGTGCCCCGTCTTC (SEQ ID NO: 388) R27nsa228R GCGCACGTCGGGGGCGAASNNCTCGGTGGGTGCCCCGTC (SEQ ID NO: 389) F28nsa229R CCAGCGCACGTCGGGGGCSNNCCGCTCGGTGGGTGCCCC (SEQ ID NO: 390) A29nsa230R GGTCCAGCGCACGTCGGGSNNGAACCGCTCGGTGGGTGC (SEQ ID NO: 391) P30nsa231R ACCGGTCCAGCGCACGTCSNNGGCGAACCGCTCGGTGGG (SEQ ID NO: 392) D31nsa232R CACACCGGTCCAGCGCACSNNGGGGGCGAACCGCTCGGT (SEQ ID NO: 393) V32nsa233R CAGCACACCGGTCCAGCGSNNGTCGGGGGCGAACCGCTC (SEQ ID NO: 394) R33nsa234R GGCCAGCACACCGGTCCASNNCACGTCGGGGGCGAACCG (SEQ ID NO: 395) W34nsa235R CTGGGCCAGCACACCGGTSNNGCGCACGTCGGGGGCGAA (SEQ ID NO: 396) T35nsa236R CTGCTGGGCCAGCACACCSNNCCAGCGCACGTCGGGGGC (SEQ ID NO: 397) G36nsa237R GAGCTGCTGGGCCAGCACSNNGGTCCAGCGCACGTCGGG (SEQ ID NO: 398) V37nsa238R TCCGAGCTGCTGGGCCAGSNNACCGGTCCAGCGCACGTC (SEQ ID NO: 399) L38nsa239R CGCTCCGAGCTGCTGGGCSNNCACACCGGTCCAGCGCAC (SEQ ID NO: 400) A39nsa240R GTCCGCTCCGAGCTGCTGSNNCAGCACACCGGTCCAGCG (SEQ ID NO: 401) Q40nsa241R GAAGTCCGCTCCGAGCTGSNNGGCCAGCACACCGGTCCA (SEQ ID NO: 402) Q41nsa242R CTCGAAGTCCGCTCCGAGSNNCTGGGCCAGCACACCGGT (SEQ ID NO: 403) L42nsa243R CACCTCGAAGTCCGCTCCSNNCTGCTGGGCCAGCACACC (SEQ ID NO: 404) G43nsa244R GATCACCTCGAAGTCCGCSNNGAGCTGCTGGGCCAGCAC (SEQ ID NO: 405) A44nsa245R CTCGATCACCTCGAAGTCSNNTCCGAGCTGCTGGGCCAG (SEQ ID NO: 406) D45nsa246R CTCCTCGATCACCTCGAASNNCGCTCCGAGCTGCTGGGC (SEQ ID NO: 407) F46nsa247R TCCCTCCTCGATCACCTCSNNGTCCGCTCCGAGCTGCTG (SEQ ID NO: 408) E47nsa248R CAGTCCCTCCTCGATCACSNNGAAGTCCGCTCCGAGCTG (SEQ ID NO: 409) V48nsa249R GCTCAGTCCCTCCTCGATSNNCTCGAAGTCCGCTCCGAG (SEQ ID NO: 410) I49nsa250R CGCGCTCAGTCCCTCCTCSNNCACCTCGAAGTCCGCTCC (SEQ ID NO: 411) E50nsa251R GCGCGCGCTCAGTCCCTCSNNGATCACCTCGAAGTCCGC (SEQ ID NO: 412) E51nsa252R GGTGCGCGCGCTCAGTCCSNNCTCGATCACCTCGAAGTC (SEQ ID NO: 413) G52nsa253R GGTGGTGCGCGCGCTCAGSNNCTCCTCGATCACCTCGAA (SEQ ID NO: 414) L53nsa193R GTTGGTGGTGCGCGCGCTSNNTCCCTCCTCGATCACCTC (SEQ ID NO: 415) S54nsa173R GATGTTGGTGGTGCGCGCSNNCAGTCCCTCCTCGATCAC (SEQ ID NO: 416) A55nsa174R GTCGATGTTGGTGGTGCGSNNGCTCAGTCCCTCCTCGAT (SEQ ID NO: 417) R56nsa257R GTCGTCGATGTTGGTGGTSNNCGCGCTCAGTCCCTCCTC (SEQ ID NO: 418) T57nsa258R GGGGTCGTCGATGTTGGTSNNGCGCGCGCTCAGTCCCTC (SEQ ID NO: 419) T58nsa259R GGTGGGGTCGTCGATGTTSNNGGTGCGCGCGCTCAGTCC (SEQ ID NO: 420) N59nsa260R ATCGGTGGGGTCGTCGATSNNGGTGGTGCGCGCGCTCAG (SEQ ID NO: 421) I60nsa261R CGGATCGGTGGGGTCGTCSNNGTTGGTGGTGCGCGCGCT (SEQ ID NO: 422) D61nsa262R CCGCGGATCGGTGGGGTCSNNGATGTTGGTGGTGCGCGC (SEQ ID NO: 423) D62nsa263R GAGCCGCGGATCGGTGGGSNNGTCGATGTTGGTGGTGCG (SEQ ID NO: 424) P63nsa264R GTTGAGCCGCGGATCGGTSNNGTCGTCGATGTTGGTGGT (SEQ ID NO: 425) T64nsa194R GCCGTTGAGCCGCGGATCSNNGGGGTCGTCGATGTTGGT (SEQ ID NO: 426) D65nsa195R CGCGCCGTTGAGCCGCGGSNNGGTGGGGTCGTCGATGTT (SEQ ID NO: 427) P66nsa267R GCTCGCGCCGTTGAGCCGSNNATCGGTGGGGTCGTCGAT (SEQ ID NO: 428) R67nsa196R GTAGCTCGCGCCGTTGAGSNNCGGATCGGTGGGGTCGTC (SEQ ID NO: 429) L68nsa269R CAGGTAGCTCGCGCCGTTSNNCCGCGGATCGGTGGGGTC (SEQ ID NO: 430) N69nsa270R CGGCAGGTAGCTCGCGCCSNNGAGCCGCGGATCGGTGGG (SEQ ID NO: 431) G70nsa271R CGACGGCAGGTAGCTCGCSNNGTTGAGCCGCGGATCGGT (SEQ ID NO: 432) A71nsa272R GCACGACGGCAGGTAGCTSNNGCCGTTGAGCCGCGGATC (SEQ ID NO: 433) S72nsa273R GAGGCACGACGGCAGGTASNNCGCGCCGTTGAGCCGCGG (SEQ ID NO: 434) Y73nsa274R CGCGAGGCACGACGGCAGSNNGCTCGCGCCGTTGAGCCG (SEQ ID NO: 435) L74nsa275R CGTCGCGAGGCACGACGGSNNGTAGCTCGCGCCGTTGAG (SEQ ID NO: 436) P75nsa276R GTGCGTCGCGAGGCACGASNNCAGGTAGCTCGCGCCGTT (SEQ ID NO: 437) S76nsa277R CAGGTGCGTCGCGAGGCASNNCGGCAGGTAGCTCGCGCC (SEQ ID NO: 438) C77nsa278R CGGCAGGTGCGTCGCGAGSNNCGACGGCAGGTAGCTCGC (SEQ ID NO: 439) L78nsa279R GAGCGGCAGGTGCGTCGCSNNGCACGACGGCAGGTAGCT (SEQ ID NO: 440) A79nsa280R GTCGAGCGGCAGGTGCGTSNNGAGGCACGACGGCAGGTA (SEQ ID NO: 441) T80nsa281R CAGGTCGAGCGGCAGGTGSNNCGCGAGGCACGACGGCAG (SEQ ID NO: 442) H81nsa282R CACCAGGTCGAGCGGCAGSNNCGTCGCGAGGCACGACGG (SEQ ID NO: 443) L82nsa283R GATCACCAGGTCGAGCGGSNNGTGCGTCGCGAGGCACGA (SEQ ID NO: 444) P83nsa284R GATGATCACCAGGTCGAGSNNCAGGTGCGTCGCGAGGCA (SEQ ID NO: 445) L84nsa285R CATGATGATCACCAGGTCSNNCGGCAGGTGCGTCGCGAG (SEQ ID NO: 446) D85nsa286R CAGCATGATGATCACCAGSNNGAGCGGCAGGTGCGTCGC (SEQ ID NO: 447) L86nsa287R GCCCAGCATGATGATCACSNNGTCGAGCGGCAGGTGCGT (SEQ ID NO: 448) V87nsa288R GGTGCCCAGCATGATGATSNNCAGGTCGAGCGGCAGGTG (SEQ ID NO: 449) I88nsa289R GTTGGTGCCCAGCATGATSNNCACCAGGTCGAGCGGCAG (SEQ ID NO: 450) I89nsa290R GTCGTTGGTGCCCAGCATSNNGATCACCAGGTCGAGCGG (SEQ ID NO: 451) M90nsa291R GGTGTCGTTGGTGCCCAGSNNGATGATCACCAGGTCGAG (SEQ ID NO: 452) L91nsa292R CTTGGTGTCGTTGGTGCCSNNCATGATGATCACCAGGTC (SEQ ID NO: 453) G92nsa293R GGCCTTGGTGTCGTTGGTSNNCAGCATGATGATCACCAG (SEQ ID NO: 454) T93nsa294R GTAGGCCTTGGTGTCGTTSNNGCCCAGCATGATGATCAC (SEQ ID NO: 455) N94nsa175R GAAGTAGGCCTTGGTGTCSNNGGTGCCCAGCATGATGAT (SEQ ID NO: 456) D95nsa197R CCGGAAGTAGGCCTTGGTSNNGTTGGTGCCCAGCATGAT (SEQ ID NO: 457) T96nsa297R GCGCCGGAAGTAGGCCTTSNNGTCGTTGGTGCCCAGCAT (SEQ ID NO: 458) K97nsa176R GGTGCGCCGGAAGTAGGCSNNGGTGTCGTTGGTGCCCAG (SEQ ID NO: 459) A98nsa299R CGGGGTGCGCCGGAAGTASNNCTTGGTGTCGTTGGTGCC (SEQ ID NO: 460) Y99nsa177R GAGCGGGGTGCGCCGGAASNNGGCCTTGGTGTCGTTGGT (SEQ ID NO: 461) F100nsa301R GTCGAGCGGGGTGCGCCGSNNGTAGGCCTTGGTGTCGTT (SEQ ID NO: 462) R101nsa302R GATGTCGAGCGGGGTGCGSNNGAAGTAGGCCTTGGTGTC (SEQ ID NO: 463) R102nsa303R CGCGATGTCGAGCGGGGTSNNCCGGAAGTAGGCCTTGGT (SEQ ID NO: 464) T103nsa304R CAGCGCGATGTCGAGCGGSNNGCGCCGGAAGTAGGCCTT (SEQ ID NO: 465) P104nsa305R GCCCAGCGCGATGTCGAGSNNGGTGCGCCGGAAGTAGGC (SEQ ID NO: 466) L105nsa306R CATGCCCAGCGCGATGTCSNNCGGGGTGCGCCGGAAGTA (SEQ ID NO: 467) D106nsa307R CGACATGCCCAGCGCGATSNNGAGCGGGGTGCGCCGGAA (SEQ ID NO: 468) I107nsa308R CACCGACATGCCCAGCGCSNNGTCGAGCGGGGTGCGCCG (SEQ ID NO: 469) A108nsa309R GAGCACCGACATGCCCAGSNNGATGTCGAGCGGGGTGCG (SEQ ID NO: 470) L109nsa310R GACGAGCACCGACATGCCSNNCGCGATGTCGAGCGGGGT (SEQ ID NO: 471) G110nsa311R CGTGACGAGCACCGACATSNNCAGCGCGATGTCGAGCGG (SEQ ID NO: 472) M111nsa312R CTGCGTGACGAGCACCGASNNGCCCAGCGCGATGTCGAG (SEQ ID NO: 473) S112nsa313R CACCTGCGTGACGAGCACSNNCATGCCCAGCGCGATGTC (SEQ ID NO: 474) V113nsa314R GAGCACCTGCGTGACGAGSNNCGACATGCCCAGCGCGAT (SEQ ID NO: 475) L114nsa315R GGTGAGCACCTGCGTGACSNNCACCGACATGCCCAGCGC (SEQ ID NO: 476) V115nsa316R GCTGGTGAGCACCTGCGTSNNGAGCACCGACATGCCCAG (SEQ ID NO: 477) T116nsa317R CGCGCTGGTGAGCACCTGSNNGACGAGCACCGACATGCC (SEQ ID NO: 478) Q117nsa318R GCCCGCGCTGGTGAGCACSNNCGTGACGAGCACCGACAT (SEQ ID NO: 479) V118nsa319R GCCGCCCGCGCTGGTGAGSNNCTGCGTGACGAGCACCGA (SEQ ID NO: 480) L119nsa320R GACGCCGCCCGCGCTGGTSNNCACCTGCGTGACGAGCAC (SEQ ID NO: 481) T120nsa321R GCCGACGCCGCCCGCGCTSNNGAGCACCTGCGTGACGAG (SEQ ID NO: 482) S121nsa322R GGTGCCGACGCCGCCCGCSNNGGTGAGCACCTGCGTGAC (SEQ ID NO: 483) A122nsa323R CGTGGTGCCGACGCCGCCSNNGCTGGTGAGCACCTGCGT (SEQ ID NO: 484) G123nsa324R GTACGTGGTGCCGACGCCSNNCGCGCTGGTGAGCACCTG (SEQ ID NO: 485) G124nsa325R CGGGTACGTGGTGCCGACSNNGCCCGCGCTGGTGAGCAC (SEQ ID NO: 486) V125nsa198R TGCCGGGTACGTGGTGCCSNNGCCGCCCGCGCTGGTGAG (SEQ ID NO: 487) G126nsa327R GGGTGCCGGGTACGTGGTSNNGACGCCGCCCGCGCTGGT (SEQ ID NO: 488) T127nsa328R CTTGGGTGCCGGGTACGTSNNGCCGACGCCGCCCGCGCT (SEQ ID NO: 489) T128nsa329R CACCTTGGGTGCCGGGTASNNGGTGCCGACGCCGCCCGC (SEQ ID NO: 490) Y129nsa330R CAGCACCTTGGGTGCCGGSNNCGTGGTGCCGACGCCGCC (SEQ ID NO: 491) P130nsa331R CACCAGCACCTTGGGTGCSNNGTACGTGGTGCCGACGCC (SEQ ID NO: 492) A131nsa332R GACCACCAGCACCTTGGGSNNCGGGTACGTGGTGCCGAC (SEQ ID NO: 493) P132nsa333R CGAGACCACCAGCACCTTSNNTGCCGGGTACGTGGTGCC (SEQ ID NO: 494) K133nsa334R CGGCGAGACCACCAGCACSNNGGGTGCCGGGTACGTGGT (SEQ ID NO: 495) V134nsa335R TGGCGGCGAGACCACCAGSNNCTTGGGTGCCGGGTACGT (SEQ ID NO: 496) L135nsa336R CGGTGGCGGCGAGACCACSNNCACCTTGGGTGCCGGGTA (SEQ ID NO: 497) V136nsa337R CAGCGGTGGCGGCGAGACSNNCAGCACCTTGGGTGCCGG (SEQ ID NO: 498) V137nsa338R CGCCAGCGGTGGCGGCGASNNCACCAGCACCTTGGGTGC (SEQ ID NO: 499) S138nsa339R GGGCGCCAGCGGTGGCGGSNNGACCACCAGCACCTTGGG (SEQ ID NO: 500) P139nsa340R CATGGGCGCCAGCGGTGGSNNCGAGACCACCAGCACCTT (SEQ ID NO: 501) P140nsa341R CGGCATGGGCGCCAGCGGSNNCGGCGAGACCACCAGCAC (SEQ ID NO: 502) P141nsa342R GTGCGGCATGGGCGCCAGSNNTGGCGGCGAGACCACCAG (SEQ ID NO: 503) L142nsa343R GGGGTGCGGCATGGGCGCSNNCGGTGGCGGCGAGACCAC (SEQ ID NO: 504) A143nsa344R CCAGGGGTGCGGCATGGGSNNCAGCGGTGGCGGCGAGAC (SEQ ID NO: 505) P144nsa345R GAACCAGGGGTGCGGCATSNNCGCCAGCGGTGGCGGCGA (SEQ ID NO: 506) M145nsa346R CTGGAACCAGGGGTGCGGSNNGGGCGCCAGCGGTGGCGG (SEQ ID NO: 507) P146nsa178R CAACTGGAACCAGGGGTGSNNCATGGGCGCCAGCGGTGG (SEQ ID NO: 508) H147nsa348R GATCAACTGGAACCAGGGSNNCGGCATGGGCGCCAGCGG (SEQ ID NO: 509) P148nsa199R GAAGATCAACTGGAACCASNNGTGCGGCATGGGCGCCAG (SEQ ID NO: 510) W149nsa179R CTCGAAGATCAACTGGAASNNGGGGTGCGGCATGGGCGC (SEQ ID NO: 511) F150nsa180R GCCCTCGAAGATCAACTGSNNCCAGGGGTGCGGCATGGG (SEQ ID NO: 512) Q151nsa352R GCCGCCCTCGAAGATCAASNNGAACCAGGGGTGCGGCAT (SEQ ID NO: 513) L152nsa353R CTCGCCGCCCTCGAAGATSNNCTGGAACCAGGGGTGCGG (SEQ ID NO: 514) I153nsa200R CTGCTCGCCGCCCTCGAASNNCAACTGGAACCAGGGGTG (SEQ ID NO: 515) F154nsa201R CTTCTGCTCGCCGCCCTCSNNGATCAACTGGAACCAGGG (SEQ ID NO: 516) E155nsa356R GGTCTTCTGCTCGCCGCCSNNGAAGATCAACTGGAACCA (SEQ ID NO: 517) G156nsa357R AGTGGTCTTCTGCTCGCCSNNCTCGAAGATCAACTGGAA (SEQ ID NO: 518) G157nsa358R CTCAGTGGTCTTCTGCTCSNNGCCCTCGAAGATCAACTG (SEQ ID NO: 519) E158nsa359R GAGCTCAGTGGTCTTCTGSNNGCCGCCCTCGAAGATCAA (SEQ ID NO: 520) Q159nsa360R GGCGAGCTCAGTGGTCTTSNNCTCGCCGCCCTCGAAGAT (SEQ ID NO: 521) K160nsa361R GCGGGCGAGCTCAGTGGTSNNCTGCTCGCCGCCCTCGAA (SEQ ID NO: 522) T161nsa362R CACGCGGGCGAGCTCAGTSNNCTTCTGCTCGCCGCCCTC (SEQ ID NO: 523) T162nsa363R GTACACGCGGGCGAGCTCSNNGGTCTTCTGCTCGCCGCC (SEQ ID NO: 524) E163nsa364R GCTGTACACGCGGGCGAGSNNAGTGGTCTTCTGCTCGCC (SEQ ID NO: 525) L164nsa365R CGCGCTGTACACGCGGGCSNNCTCAGTGGTCTTCTGCTC (SEQ ID NO: 526) A165nsa366R GAGCGCGCTGTACACGCGSNNGAGCTCAGTGGTCTTCTG (SEQ ID NO: 527) R166nsa367R CGCGAGCGCGCTGTACACSNNGGCGAGCTCAGTGGTCTT (SEQ ID NO: 528) V167nsa368R CGACGCGAGCGCGCTGTASNNGCGGGCGAGCTCAGTGGT (SEQ ID NO: 529) Y168nsa369R GAACGACGCGAGCGCGCTSNNCACGCGGGCGAGCTCAGT (SEQ ID NO: 530) S169nsa370R CATGAACGACGCGAGCGCSNNGTACACGCGGGCGAGCTC (SEQ ID NO: 531) A170nsa371R CTTCATGAACGACGCGAGSNNGCTGTACACGCGGGCGAG (SEQ ID NO: 532) L171nsa372R CACCTTCATGAACGACGCSNNCGCGCTGTACACGCGGGC (SEQ ID NO: 533) A172nsa373R CGGCACCTTCATGAACGASNNGAGCGCGCTGTACACGCG (SEQ ID NO: 534) S173nsa374R GAACGGCACCTTCATGAASNNCGCGAGCGCGCTGTACAC (SEQ ID NO: 535) F174nsa375R GAAGAACGGCACCTTCATSNNCGACGCGAGCGCGCTGTA (SEQ ID NO: 536) M175nsa376R GTCGAAGAACGGCACCTTSNNGAACGACGCGAGCGCGCT (SEQ ID NO: 537) K176nsa377R CGCGTCGAAGAACGGCACSNNCATGAACGACGCGAGCGC (SEQ ID NO: 538) V177nsa378R ACCCGCGTCGAAGAACGGSNNCTTCATGAACGACGCGAG (SEQ ID NO: 539) P178nsa379R CGAACCCGCGTCGAAGAASNNCACCTTCATGAACGACGC (SEQ ID NO: 540) F179nsa380R CACCGAACCCGCGTCGAASNNCGGCACCTTCATGAACGA (SEQ ID NO: 541) F180nsa381R GATCACCGAACCCGCGTCSNNGAACGGCACCTTCATGAA (SEQ ID NO: 542) D181nsa382R GCTGATCACCGAACCCGCSNNGAAGAACGGCACCTTCAT (SEQ ID NO: 543) A182nsa383R GGTGCTGATCACCGAACCSNNGTCGAAGAACGGCACCTT (SEQ ID NO: 544) G183nsa384R GTCGGTGCTGATCACCGASNNCGCGTCGAAGAACGGCAC (SEQ ID NO: 545) S184nsa385R GCCGTCGGTGCTGATCACSNNACCCGCGTCGAAGAACGG (SEQ ID NO: 546) V185nsa386R GACGCCGTCGGTGCTGATSNNCGAACCCGCGTCGAAGAA (SEQ ID NO: 547) I186nsa387R GTCGACGCCGTCGGTGCTSNNCACCGAACCCGCGTCGAA (SEQ ID NO: 548) S187nsa388R TCCGTCGACGCCGTCGGTSNNGATCACCGAACCCGCGTC (SEQ ID NO: 549) T188nsa389R GATTCCGTCGACGCCGTCSNNGCTGATCACCGAACCCGC (SEQ ID NO: 550) D189nsa390R GTGGATTCCGTCGACGCCSNNGGTGCTGATCACCGAACC (SEQ ID NO: 551) G190nsa391R GAAGTGGATTCCGTCGACSNNGTCGGTGCTGATCACCGA (SEQ ID NO: 552) V191nsa392R GGTGAAGTGGATTCCGTCSNNGCCGTCGGTGCTGATCAC (SEQ ID NO: 553) D192nsa393R CTCGGTGAAGTGGATTCCSNNGACGCCGTCGGTGCTGAT (SEQ ID NO: 554) G193nsa394R GGCCTCGGTGAAGTGGATSNNGTCGACGCCGTCGGTGCT (SEQ ID NO: 555) I194nsa181R GTTGGCCTCGGTGAAGTGSNNTCCGTCGACGCCGTCGGT (SEQ ID NO: 556) H195nsa396R ATTGTTGGCCTCGGTGAASNNGATTCCGTCGACGCCGTC (SEQ ID NO: 557) F196nsa182R GCGATTGTTGGCCTCGGTSNNGTGGATTCCGTCGACGCC (SEQ ID NO: 558) T197nsa398R ATCGCGATTGTTGGCCTCSNNGAAGTGGATTCCGTCGAC (SEQ ID NO: 559) E198nsa399R GAGATCGCGATTGTTGGCSNNGGTGAAGTGGATTCCGTC (SEQ ID NO: 560) A199nsa400R CCCGAGATCGCGATTGTTSNNCTCGGTGAAGTGGATTCC (SEQ ID NO: 561) N200nsa401R CACCCCGAGATCGCGATTSNNGGCCTCGGTGAAGTGGAT (SEQ ID NO: 562) N201nsa402R GGCCACCCCGAGATCGCGSNNGTTGGCCTCGGTGAAGTG (SEQ ID NO: 563) R202nsa403R GAGGGCCACCCCGAGATCSNNATTGTTGGCCTCGGTGAA (SEQ ID NO: 564) D203nsa404R CGCGAGGGCCACCCCGAGSNNGCGATTGTTGGCCTCGGT (SEQ ID NO: 565) L204nsa405R TTCCGCGAGGGCCACCCCSNNATCGCGATTGTTGGCCTC (SEQ ID NO: 566) G205nsa406R CTGTTCCGCGAGGGCCACSNNGAGATCGCGATTGTTGGC (SEQ ID NO: 567) V206nsa407R CACCTGTTCCGCGAGGGCSNNCCCGAGATCGCGATTGTT (SEQ ID NO: 568) A207nsa408R CTGCACCTGTTCCGCGAGSNNCACCCCGAGATCGCGATT (SEQ ID NO: 569) L208nsa409R GCTCTGCACCTGTTCCGCSNNGGCCACCCCGAGATCGCG (SEQ ID NO: 570) A209nsa410R CAGGCTCTGCACCTGTTCSNNGAGGGCCACCCCGAGATC (SEQ ID NO: 571) E210nsa411R CAGCAGGCTCTGCACCTGSNNCGCGAGGGCCACCCCGAG (SEQ ID NO: 572) Q211nsa412R TTACAGCAGGCTCTGCACSNNTTCCGCGAGGGCCACCCC (SEQ ID NO: 573) V212nsa413R CTTTTACAGCAGGCTCTGSNNCTGTTCCGCGAGGGCCAC (SEQ ID NO: 574) Q213nsa414R GCCCTTTTACAGCAGGCTSNNCACCTGTTCCGCGAGGGC (SEQ ID NO: 575) S214nsa415R TTCGCCCTTTTACAGCAGSNNCTGCACCTGTTCCGCGAG (SEQ ID NO: 576) L215nsa416R GAATTCGCCCTTTTACAGSNNGCTCTGCACCTGTTCCGC (SEQ ID NO: 577) L216nsa417R GCAGAATTCGCCCTTTTASNNCAGGCTCTGCACCTGTTC (SEQ ID NO: 578)QC Method to Create Site-Saturation Libraries

The QC reaction consisted of 40.25 μL of sterile distilled H₂O, 5 μL ofPfuTurbo 10× buffer from the kit, 1 μL dNTPs from the kit, 1.25 μL offorward primer (100 ng/μL), 1.25 μL reverse primer (100 ng/μL), 0.25 μLof pMSAT-NcoI miniprep DNA as template (˜50 ng), and 1 μL of PfuTurbofrom the kit, for a total of 50 μL. The cycling conditions were 95° C.for 1 min, once, followed by 19-20 cycles of 95° C. for 30 to 45 sec,55° C. for 1 min, and 68° C. for 5 to 8 min. To analyze the reaction, 5μL of the reaction was run on a 0.8% E-gel (Invitrogen) upon completion.Next, DpnI digestion was carried out twice sequentially, with 1 μL and0.5 μL of enzyme at 37° C. for 2 to 8 hours. A negative control wascarried out under similar conditions, but without any primers. Then, 1μL of the DpnI-digested reaction product was transformed into 50 μL ofone-shot TOP10 electrocompetent cells (Invitrogen) using a BioRadelectroporator. Then, 300 μL of SOC provided with the TOP10 cells(Invitrogen) were added to the electroporated cells and incubated withshaking for 1 hour before plating on LA plates containing 10 ppmkanamycin. The plates were incubated at 37° C. overnight. After thisincubation, 96 colonies from each of the libraries (i.e., each site)were inoculated in 200 μL of LB containing 10-50 ppm of kanamycin in96-well microtiter plates. The plates were frozen at −80° C. afteraddition of glycerol to 20% final concentration, and they were used forhigh throughput sequencing at Genaissance with the M13F and M13Rprimers.

QCMS Method to Create Site-Saturation Libraries

The QCMS reaction consisted of 19.25 μL of sterile distilled H₂O, 2.5 μLof 10× buffer from the kit, 1 μL dNTPs from the kit, 1 μL of 5′phosphorylated forward primer (100 ng/μL), 0.25 μL of pMSAT-NcoIminiprep DNA as template (˜50 ng), and 1 μL of the enzyme blend from thekit for a total of 25 μL. The cycling conditions were 95° C. for 1 minonce, followed by 30 cycles of 95° C. for 1 min, 55° C. for 1 min, and68° C. for 8 min. To analyze the reaction product, 5 μL of the reactionwere run on a 0.8% E-gel (Invitrogen) upon completion. Next, DpnIdigestion was carried out twice sequentially, with 0.5 μL of enzyme at37° C. for 2 to 8 hours. The controls, transformation, and sequencingwas performed as for the QC method described above.

Details of Screening Plate Preparation

Using a sterilized stamping tool with 96 pins, the frozen clones fromeach sequenced library plate were stamped on to a large LA platecontaining 10 ppm kanamycin. The plate was then incubated overnight at37° C. Individual mutant clones each representing each one of the 19substitutions (or as many that were obtained) were inoculated into aCostar 96-well plate containing 195 μL of LB made with 2 fold greateryeast extract and 10 ppm kanamycin. Each mutant clone for a given sitewas inoculated in quadruplicate. The plate was grown at 37° C. and 225rpm shaking for 18 hrs in a humidified chamber. In a separate 96-wellplate, 26 μL of BugBuster (Novagen) with DNase were added to each well.Next, 125 μL of the library clone cultures were added to theBugBuster-containing plate in corresponding wells and the plate wasfrozen at 80° C. The plate was thawed, frozen and thawed again beforeuse of the lysates in the peracid formation and peracid hydrolysisassays described herein.

Combinatorial Libraries and Mutants

From the screening of the single site-saturation libraries, theimportant sites and substitutions were identified and combined indifferent combinatorial libraries. For example, libraries described inTable 8-3 were created using the following sites and substitutions:

L12C, Q, G

T25S, G, P

L53H, Q, G, S

S54V, L, A, P, T, R

A55G, T

R67T, Q, N, G, E, L, F

K97R

V125S, G, R, A, P

F154Y

F196G

TABLE 8-3 Libraries Parent Library Description Template Method NSAA1L12G S54(NNS) L12G QC NSAA2 S54V L12(NNS) S54V QC NSAA3 L12(NNS)S54(NNS) WT QCMS NSAB1 S54V T25(NNS) S54V QC NSAB2 S54V R67(NNS) S54V QCNSAB3 S54V V125(NNS) S54V QC NSAB4 L12I S54V T25(NNS) L12I S54V QC NSAB5L12I S54V R67(NNS) L12I S54V QC NSAB6 L12I S54V V125(NNS) L12I S54V QCNSAC1 S54(NNS) R67(NNS) WT QCMS V125(NNS) NSAC2 43 primer library; 10sites S54V QCMS (100 ng total primers) NSAC3 same as nsaC2 but 300 ngS54V QCMS total primers NSAC4 32 primer library, 8 sites S54V QCMS (100ng total primers) NSAC5 same as nsaC4 but 300 ng S54V QCMS total primersNSAC6 8 primers, 7 substitutions, S54V QCMS 5 sites (100 ng totalprimers) NSAC7 same as nsaC6 but 300 ng S54V QCMS total primers *NNSindicates site-saturation library **All parent templates were derivedfrom the pMSAT-NcoI plasmid and contained mutations at the indicatedcodons with in the M. smegmatis perhydrolase gene

The QC or QCMS methods were used to create the combinations. The QCreaction was carried out as described above, with the exception beingthe template plasmid, which consisted of 0.25 μL of miniprep DNA of theL12G mutant, S54V mutant, or the L12I S54V double mutant plasmid derivedfrom pMSAT-NcoI. The QCMS reaction was also carried out as describedabove, with the exception of template and primers. In this case, 0.25 μLof the pMSAT-NcoI template were used for NSAC1 and NSAA3 or S54Vtemplate for NSAC2-C7 libraries. The NSAA3 and the NSAC1 libraries weremade using 100 ng of each of the primers shown in the Table 8-4. TheNSAC2, NSAC4, and NSAC6 libraries were made with a total of 100 ng ofall primers (all primers being equimolar), and NSAC3, NSAC5, NSAC7libraries were made with a total of 300 ng of all primers (all primersbeing approximately equimolar)

TABLE 8-4 Libraries Primer Libraries Name Primer Sequence NSAC1S54NNS-FP gtgatcgaggagggactgnnsgcgcgcaccaccaacatc (SEQ ID NO: 579) NSAC1R67NNS-FP acgaccccaccgatccgnnsctcaacggcgcgagctac (SEQ ID NO: 580) NSAC1V125NNS- ctcaccagcgcgggcggcnnsggcaccacgtacccggca (SEQ ID NO: 581) FPNSAC2- L12C ctgtgtttcggtgattccTGCacctggggctgggtcccc (SEQ ID NO: 582) C5NSAC2- L12Q ctgtgtttcggtgattccCAGacctggggctgggtcccc (SEQ ID NO: 583) C7NSAC2- L12I ctgtgtttcggtgattccATCacctggggctgggtcccc (SEQ ID NO: 584) C5NSAC2- L12M ctgtgtttcggtgattccATGacctggggctgggtcccc (SEQ ID NO: 585) C3NSAC2- L12T ctgtgtttcggtgattccACGacctggggctgggtcccc (SEQ ID NO: 586) C3NSA2- T25S gtcgaagacggggcacccAGCgagcggttcgcccccgac (SEQ ID NO: 587) C5NSAC2- T25G gtcgaagacggggcacccGGCgagcggttcgcccccgac (SEQ ID NO: 588) C5NSAC2- T25P gtcgaagacggggcacccCCGgagcggttcgcccccgac (SEQ ID NO: 589) C3NSAC2- L53H gaggtgatcgaggagggaCACagcgcgcgcaccaccaac (SEQ ID NO: 590) C7NSAC2- L53Q gaggtgatcgaggagggaCAGagcgcgcgcaccaccaac (SEQ ID NO: 591) C3NSAC2- L53G gaggtgatcgaggagggaGGCagcgcgcgcaccaccaac (SEQ ID NO: 592) C3NSAC2- L53S gaggtgatcgaggagggaAGCagcgcgcgcaccaccaac (SEQ ID NO: 593) C3NSAC2- L53HS54V gaggtgatcgaggagggaCACGTGgcgcgcaccaccaac (SEQ ID C7 NO:594) NSAC2- L53QS54V gaggtgatcgaggagggaCAGGTGgcgcgcaccaccaac (SEQ ID C3NO: 595) NSAC2- L53GS54V gaggtgatcgaggagggaGGCGTGgcgcgcaccaccaac (SEQ IDC3 NO: 596) NSAC2- L53SS54V gaggtgatcgaggagggaAGCGTGgcgcgcaccaccaac (SEQID C3 NO: 597) NSAC2- S54V gtgatcgaggagggactgGTGgcgcgcaccaccaacatc (SEQID NO: 598) C7 NSAC2- S54L gtgatcgaggagggactgCTGgcgcgcaccaccaacatc (SEQID NO: 599) C5 NSAC2- A55G atcgaggagggactgagcGGCcgcaccaccaacatcgac (SEQID NO: 600) C5 NSAC2- A55T atcgaggagggactgagcACGcgcaccaccaacatcgac (SEQID NO: 601) C5 NSAC2- A55GS54V atcgaggagggactgGTGGGCcgcaccaccaacatcgac(SEQ ID NO: 602) C5 NSAC2- A55TS54VatcgaggagggactgGTGACGcgcaccaccaacatcgac (SEQ ID NO: 603) C5 NSAC2- R67TgacgaccccaccgatccgACGctcaacggcgcgagctac (SEQ ID NO: 604) C5 NSAC2- R67QgacgaccccaccgatccgCAGctcaacggcgcgagctac (SEQ ID NO: 605) C5 NSAC2- R67NgacgaccccaccgatccgAACctcaacggcgcgagctac (SEQ ID NO: 606) C7 NSAC2- K97RctgggcaccaacgacaccCGCgcctacttccggcgcacc (SEQ ID NO: 607) C5 NSAC2- V125SctcaccagcgcgggcggcAGCggcaccacgtacccggca (SEQ ID NO: 608) C5 NSAC2- V125GctcaccagcgcgggcggcGGCggcaccacgtacccggca (SEQ ID NO: 609) C7 NSAC2- V125RctcaccagcgcgggcggcCGCggcaccacgtacccggca (SEQ ID NO: 610) C5 NSAC2- V125ActcaccagcgcgggcggcGCGggcaccacgtacccggca (SEQ ID NO: 611) C5 NSAC2- V125PctcaccagcgcgggcggcCCGggcaccacgtacccggca (SEQ ID NO: 612) C5 NSAC2- F154YccctggttccagttgatcTACgagggcggcgagcagaag (SEQ ID NO: 613) C3 NSAC2- F196GggcgtcgacggaatccacGGCaccgaggccaacaatcgc (SEQ ID NO: 614) C3 NSAC2-R67G-re gacgaccccaccgatccgGGCctcaacggcgcgagctac (SEQ ID NO: 615) C7NSAC2- R67E-re gacgaccccaccgatccgGAGctcaacggcgcgagctac (SEQ ID NO: 616)C5 NSAC2- R67F-re gacgaccccaccgatccgTTCctcaacggcgcgagctac (SEQ ID NO:617) C5 NSAC2- R67L-re gacgaccccaccgatccgCTGctcaacggcgcgagctac (SEQ IDNO: 618) C5 NSAC2- S54P gtgatcgaggagggactgCCGgcgcgcaccaccaacatc (SEQ IDNO: 619) C5 NSAC2- S54R gtgatcgaggagggactgCGCgcgcgcaccaccaacatc (SEQ IDNO: 620) C5 NSAC2- S54G gtgatcgaggagggactgGGCgcgcgcaccaccaacatc (SEQ IDNO: 621) C5 NSAC2- S54T gtgatcgaggagggactgACGgcgcgcaccaccaacatc (SEQ IDNO: 622) C5 NSAC2- S54I gtgatcgaggagggactgATCgcgcgcaccaccaacatc (SEQ IDNO: 623) C7 NSAC2- S54K gtgatcgaggagggactgAAGgcgcgcaccaccaacatc (SEQ IDNO: 624) C5Screening of Combinatorial Libraries and Mutants

For each of the NSAB1-B6 libraries, a 96-well plate full of clones wasfirst sequenced. Once the sequencing results were analyzed, the mutantsobtained for each library were inoculated in quadruplicate, similar tothe site-saturation libraries described above. For the NSAC1-C7libraries, 96 colonies per/plate/library were initially inoculated, andeach plate was screened without sequencing. Upon screening, somelibraries looked better than others. Several plates for each of theNSAC1, C2, C4, C6 libraries were screened. The “winners” from thesesingle isolate screening plates were then streaked out for singles ordirectly screened in quadruplicate just like the site-saturationlibraries (i.e., as described above). Only the “winners” identified weresequenced.

Example 9 Improved Properties of Multiply Mutated Perhydrolase Variants

In this Example, experiments conducted to assess the properties ofmultiply-mutated perhydrolase variants are described. In theseexperiments, combinatorial mutants obtained from combinatorial librarieswere tested in their performance in perhydrolysis, peracid hydrolysisand perhydrolysis to hydrolysis ratio. These parameters were measured inthe HPLC or ABTS assays described in Example 2, above. Combinatorialvariants tested were:

L12I S54V,

L12M S54T,

L12T S54V,

L12Q T25S S54V,

L53H S54V,

S54P V125R,

S54V V125G,

S54V F196G,

S54V K97R V125G, and

A55G R67T K97R V125G,

As is indicated in Table 9-1 below, all of these variants were betterthan wild type enzyme in at least one of the properties of interest.

TABLE 9-1 Results for Multiple Variants Fold-Improvement in PropertyMultiple Variant Perhydrolysis Peracid Hydrolysis Ratio L12I S54V 2 2.5L12M S54T 1.6 3 L12T S54V 1.5 2.5 L12Q T25S S54V 4 to 5 L53H S54V 2 4 to5 S54P V125R 4 S54V V125G 2 4 S54V F196G 2 S54V K97R V125G 2 A55G R67TK97R 1.6 4 to 5 V125G

Example 10 PAF and PAD Assays of Perhydrolase Variants

In this Example, assay results for PAF and PAD testing of perhydrolasevariants are provided. The tests were conducted as described in Example1, above. In addition, Tables are provided in which the proteinexpression of the variant was greater than wild-type under the sameculture conditions (described herein). These results are indicated asthe “protein performance index.” Thus, a number greater than “1” in theprotein performance index indicates that more protein was made for theparticular variant than the wild-type. In the following Tables, “WT”indicates the wild-type amino acid residue; “Pos” indicates the positionin the amino acid sequence; “Mut.” and “Var” indicate the amino acidresidue substituted at that particular position; “prot.” indicates“protein; and “Perf. Ind” indicates the performance index.

TABLE 10-1 PAF Assay Results PAF WT/Pos/ Perf. Position Mutation VariantInd. 3 K003Y Y 1.058244 3 K003I I 1.053242 3 K003L L 1.038686 3 K003T T1.009071 3 K003H H 1.00528 4 R004Q Q 1.025332 5 I005T T 1.12089 5 I005SS 1.023576 6 L006V V 1.072388 6 L006I I 1.066182 6 L006T T 1.062078 7C007K K 2.687956 7 C007Y Y 2.08507 7 C007I I 1.758096 7 C007H H 1.7314757 C007A A 1.423943 7 C007G G 1.393781 7 C007M M 1.126028 10 D010L L3.97014 10 D010W W 3.179778 10 D010K K 2.133852 10 D010Y Y 1.508981 10D010T T 1.473387 10 D010I I 1.281927 12 L012Q Q 2.651732 12 L012C C2.289224 12 L012A A 1.100171 15 G015A A 1.543799 15 G015S S 1.05273 17V017G G 1.173641 17 V017R R 1.09735 17 V017A A 1.012116 18 P018Y Y1.332844 18 P018N N 1.331062 18 P018C C 1.261104 18 P018E E 1.217708 18P018V V 1.185736 18 P018R R 1.16328 18 P018Q Q 1.124133 18 P018H H1.120443 18 P018G G 1.068272 19 V019G G 1.317001 19 V019S S 1.235759 19V019R R 1.025471 19 V019L L 1.002833 21 D021K K 1.062138 21 D021W W1.040173 22 G022A A 1.554264 22 G022T T 1.032118 22 G022S S 1.022133 25T025G G 1.857878 25 T025S S 1.59954 25 T025A A 1.327579 25 T025I I1.019417 26 E026M M 2.002044 26 E026A A 1.927099 26 E026R R 1.484814 26E026K K 1.464368 26 E026T T 1.441939 26 E026C C 1.403045 26 E026V V1.392881 26 E026N N 1.366419 26 E026H H 1.329562 26 E026L L 1.295378 26E026G G 1.283477 26 E026S S 1.271403 26 E026W W 1.251752 27 R027K K1.215697 28 F028M M 1.331874 28 F028A A 1.269493 28 F028W W 1.156698 28F028L L 1.08849 28 F028S S 1.046063 29 A029W W 1.912244 29 A029V V1.799733 29 A029R R 1.757225 29 A029Y Y 1.697554 29 A029G G 1.595061 29A029S S 1.486877 29 A029T T 1.424584 29 A029E E 1.115768 29 A029C C1.07522 30 P030K K 1.207673 30 P030R R 1.164892 30 P030V V 1.063047 30P030T T 1.05383 30 P030A A 1.045476 30 P030S S 1.031747 30 P030Q Q1.013468 30 P030H H 1.012332 30 P030E E 1.006761 31 D031W W 1.834044 31D031L L 1.810564 31 D031T T 1.450556 31 D031G G 1.441703 31 D031F F1.438268 31 D031N N 1.339422 31 D031V V 1.280091 31 D031A A 1.240923 31D031R R 1.222181 31 D031S S 1.152736 31 D031E E 1.132795 31 D031Q Q1.069797 32 V032K K 1.08606 32 V032R R 1.045435 33 R033S S 1.000491 36G036I I 1.320156 36 G036K K 1.265563 36 G036L L 1.237473 38 L038L L6.528092 38 L038V V 5.735873 38 L038C C 4.182031 38 L038K K 4.135067 38L038A A 3.844719 38 L038S S 2.467764 40 Q040K K 2.613726 40 Q040I I2.576806 40 Q040W W 2.394926 40 Q040L L 2.144687 40 Q040T T 2.006487 40Q040R R 1.885154 40 Q040Y Y 1.825366 40 Q040G G 1.785768 40 Q040S S1.565973 40 Q040N N 1.528677 40 Q040D D 1.16151 40 Q040E E 1.075259 41Q041K K 1.381385 41 Q041R R 1.190317 41 Q041W W 1.141041 41 Q041H H1.123719 41 Q041S S 1.107641 41 Q041Y Y 1.091652 41 Q041V V 1.070265 41Q041A A 1.032945 41 Q041L L 1.000416 42 L042K K 2.463086 42 L042W W2.056507 42 L042H H 1.917245 42 L042R R 1.378137 42 L042G G 1.172748 42L042T T 1.079826 42 L042F F 1.072948 43 G043A A 1.49082 43 G043C C1.47701 43 G043K K 1.424919 43 G043M M 1.371202 43 G043Y Y 1.262703 43G043E E 1.250311 43 G043L L 1.216516 43 G043R R 1.215829 43 G043S S1.178103 43 G043H H 1.169457 43 G043P P 1.080176 44 A044F F 2.84399 44A044V V 2.133682 44 A044C C 1.796096 44 A044L L 1.607918 44 A044W W1.395243 44 A044M M 1.199028 45 D045K K 1.342858 45 D045T T 1.268367 45D045R R 1.158768 45 D045W W 1.145157 45 D045S S 1.133098 45 D045G G1.12761 45 D045H H 1.127539 45 D045F F 1.11152 45 D045L L 1.054441 45D045V V 1.050576 45 D045Q Q 1.04498 45 D045A A 1.037993 46 F046E E1.247552 46 F046D D 1.174794 46 F046G G 1.016913 46 F046K K 1.003326 47E047R R 2.448525 47 E047T T 1.960505 47 E047P P 1.361173 47 E047S S1.278809 47 E047H H 1.266229 47 E047G G 1.197541 47 E047K K 1.19183 47E047F F 1.092281 47 E047I I 1.030029 49 I049G G 1.342918 49 I049H H1.265204 49 I049S S 1.238211 49 I049K K 1.230871 49 I049V V 1.203314 49I049L L 1.136805 49 I049Y Y 1.068104 49 I049R R 1.052285 49 I049E E1.015762 49 I049M M 1.00526 50 E050L L 1.191901 50 E050M M 1.178039 50E050A A 1.124087 51 E051V V 1.471315 51 E051A A 1.279983 51 E051G G1.217963 51 E051T T 1.182792 51 E051L L 1.112889 51 E051I I 1.072835 53L053H H 5.05321 53 L053Q Q 1.480206 53 L053G G 1.317357 53 L053S S1.161011 53 L053T T 1.019146 54 S054P P 5.198689 54 S054I I 4.775938 54S054V V 4.722033 54 S054A A 3.455902 54 S054R R 3.375793 54 S054L L2.015828 54 S054T T 1.459971 54 S054K K 1.438715 54 S054G G 1.429605 54S054C C 1.259773 54 S054Q Q 1.03365 55 A055G G 1.694814 55 A055T T1.692885 57 T057S S 1.633613 57 T057R R 1.605072 57 T057V V 1.281788 57T057I I 1.189062 59 N059W W 1.035044 59 N059R R 1.002315 60 I060H H1.02415 60 I060R R 1.003947 61 D061H H 1.439407 61 D061S S 1.259714 61D061R R 1.105425 61 D061I I 1.076937 61 D061F F 1.00566 62 D062E E1.019293 63 P063G G 1.709657 63 P063T T 1.499483 63 P063M M 1.460336 63P063S S 1.416192 63 P063K K 1.404615 63 P063A A 1.347541 63 P063Y Y1.346046 63 P063W W 1.34587 63 P063V V 1.313631 63 P063R R 1.310696 63P063F F 1.246299 63 P063L L 1.146416 63 P063Q Q 1.093179 64 T064G G1.234467 64 T064S S 1.114348 65 D065A A 1.312312 65 D065S S 1.166849 65D065H H 1.096335 66 P066R R 1.846257 66 P066V V 1.828926 66 P066H H1.589631 66 P066I I 1.588219 66 P066G G 1.499901 66 P066Q Q 1.463705 66P066T T 1.410091 66 P066S S 1.390845 66 P066Y Y 1.330685 66 P066L L1.137635 66 P066N N 1.122261 67 R067N N 1.580401 67 R067G G 1.390129 67R067T T 1.284643 67 R067F F 1.25763 67 R067L L 1.203316 67 R067Q Q1.164899 67 R067W W 1.066028 67 R067E E 1.044676 67 R067P P 1.012761 68L068E E 1.435218 68 L068W W 1.209193 68 L068I I 1.125898 68 L068G G1.092454 68 L068V V 1.088042 68 L068H H 1.051612 68 L068T T 1.032331 69N069V V 1.989028 69 N069K K 1.71908 69 N069R R 1.493163 69 N069I I1.469946 69 N069H H 1.357968 69 N069T T 1.351305 69 N069L L 1.299547 69N069S S 1.205171 69 N069G G 1.19653 69 N069Q Q 1.074622 69 N069W W1.049602 69 N069C C 1.048373 71 A071S S 1.751794 71 A071T T 1.700442 71A071H H 1.697558 71 A071G G 1.58881 71 A071I I 1.507841 71 A071E E1.445699 71 A071K K 1.441146 71 A071R R 1.401499 71 A071N N 1.232241 71A071L L 1.231991 71 A071F F 1.127538 71 A071C C 1.00977 72 S072L L1.257945 72 S072H H 1.208899 72 S072G G 1.198197 72 S072T T 1.10065 72S072V V 1.080089 72 S072Y Y 1.066178 73 Y073R R 1.2555 73 Y073Q Q1.23429 73 Y073S S 1.165683 73 Y073K K 1.070678 76 S076P P 1.229172 77C077T T 1.120603 77 C077V V 1.052586 77 C077G G 1.013806 78 L078G G4.975852 78 L078H H 4.824004 78 L078E E 3.007159 78 L078N N 2.683604 78L078T T 1.867711 78 L078Q Q 1.726942 78 L078V V 1.534239 78 L078I I1.434206 78 L078Y Y 1.387889 79 A079H H 1.927914 79 A079L L 1.796126 79A0791 I 1.592463 79 A079M M 1.499635 79 A079N N 1.475806 79 A079Q Q1.472484 79 A079R R 1.465943 79 A079W W 1.270538 79 A079T T 1.169146 79A079E E 1.123457 80 T080C C 1.310752 80 T080V V 1.230659 80 T080G G1.160318 80 T080A A 1.000722 82 L082P P 1.456374 82 L082G G 1.379439 82L082R R 1.339485 82 L082H H 1.332844 82 L082K K 1.1909 82 L082T T1.17992 82 L082I I 1.171013 82 L082S S 1.153417 82 L082V V 1.019854 83P083K K 1.369406 83 P083G G 1.313431 83 P083H H 1.265876 83 P083R R1.194464 83 P083S S 1.171208 84 L084K K 1.099089 84 L084H H 1.008187 85D085Q Q 3.093245 85 D085R R 2.379647 85 D085S S 2.284009 85 D085H H1.548556 85 D085N N 1.539497 85 D085G G 1.413812 85 D085T T 1.329395 85D085E E 1.117228 85 D085F F 1.008028 86 L086A A 1.376284 86 L086C C1.156625 86 L086G G 1.145834 95 D095E E 2.044825 96 T096S S 1.044425 97K097R R 2.798748 97 K097Q Q 1.136975 100 F100W W 1.082799 100 F100E E1.0116 101 R101K K 1.244945 103 T103W W 1.261503 103 T103Y Y 1.193299103 T103G G 1.113343 103 T103K K 1.093573 103 T103I I 1.076338 103 T103LL 1.050734 104 P104H H 2.837034 104 P104T T 2.696977 104 P104G G2.672719 104 P104V V 2.585315 104 P104S S 2.481687 104 P104I I 2.431309104 P104W W 2.051785 104 P104C C 1.951282 104 P104E E 1.837373 104 P104FF 1.785718 104 P104N N 1.624722 104 P104R R 1.618032 104 P104Q Q1.343174 104 P104M M 1.093185 105 L105P P 1.713219 105 L105C C 1.557999105 L105F F 1.295759 105 L105W W 1.283998 105 L105G G 1.078743 106 D106KK 1.278457 106 D106L L 1.198148 106 D106G G 1.178297 106 D106H H1.090134 106 D106E E 1.084931 106 D106T T 1.061622 106 D106I I 1.036191106 D106F F 1.021513 106 D106C C 1.005553 107 I107E E 2.551108 107 I107SS 2.044692 107 I107N N 1.810584 107 I107G G 1.764761 107 I107V V1.001703 108 A108L L 1.407382 108 A108T T 1.050964 109 L109N N 1.523277109 L109W W 1.296964 109 L109Q Q 1.182653 109 L109Y Y 1.155328 109 L109II 1.053129 109 L109D D 1.003394 111 M111K K 1.977248 111 M111I I1.949343 111 M111L L 1.546317 111 M111T T 1.489808 111 M111F F 1.467344111 M111V V 1.466478 111 M111Y Y 1.42589 111 M111S S 1.031939 112 S112LL 1.027928 112 S112H H 1.001485 113 V113L L 1.503622 113 V113H H1.339003 113 V113K K 1.192607 113 V113R R 1.133751 113 V113Y Y 1.113256113 V113F F 1.045057 113 V113Q Q 1.032496 115 V115W W 1.234 115 V115T T1.145757 115 V115L L 1.117398 115 V115G G 1.089596 115 V115I I 1.050387115 V115Y Y 1.032052 116 T116G G 1.095496 116 T116A A 1.006702 117 Q117HH 2.327857 117 Q117T T 2.233854 117 Q117Y Y 2.227983 117 Q117W W2.155359 117 Q117V V 2.154646 117 Q117G G 2.080223 117 Q117A A 2.048752117 Q117S S 1.949232 117 Q117F F 1.573776 117 Q117R R 1.564466 117 Q117MM 1.541944 117 Q117E E 1.145341 118 V118Y Y 1.25067 118 V118K K 1.125917118 V118G G 1.083422 120 T120S S 1.089798 121 S121L L 1.348931 121 S121WW 1.333741 121 S121R R 1.25879 121 S121K K 1.241105 121 S121G G 1.204547121 S121C C 1.177769 121 S121N N 1.143954 121 S121T T 1.132507 121 S121AA 1.120633 121 S121V V 1.120454 122 A122H H 1.137861 122 A122I I1.133601 122 A122T T 1.083131 122 A122K K 1.082552 122 A122V V 1.041449122 A122S S 1.031411 124 G124L L 1.91642 124 G124I I 1.853337 124 G124TT 1.63716 124 G124H H 1.588068 124 G124V V 1.441979 124 G124F F 1.320782124 G124S S 1.269245 124 G124Y Y 1.234423 124 G124R R 1.144212 124 G124QQ 1.123498 125 V125G G 2.948291 125 V125S S 1.942881 125 V125A A1.689696 125 V125P P 1.50166 125 V125R R 1.301534 125 V125D D 1.238852125 V125Y Y 1.080394 125 V125I I 1.010779 126 G126T T 1.577938 126 G126PP 1.171092 126 G126L L 1.169527 127 T127H H 1.57251 127 T127V V 1.073821127 T127I I 1.063668 127 T127S S 1.046984 128 T128L L 1.064623 128 T128KK 1.062947 148 P148V V 2.426937 148 P148K K 1.786508 148 P148L L1.638438 148 P148A A 1.637334 148 P148R R 1.509086 148 P148T T 1.501359148 P148Y Y 1.459512 148 P148S S 1.45564 148 P148E E 1.417449 148 P148FF 1.367568 148 P148Q Q 1.334517 148 P148D D 1.030185 150 F150L L1.290835 150 F150E E 1.228159 153 I153K K 1.618543 153 I153H H 1.464262153 I153T T 1.271928 153 I153L L 1.270149 153 I153F F 1.227821 153 I153AA 1.194659 154 F154Y Y 1.323693 196 F196H H 1.774774 196 F196L L1.768072 196 F196C C 1.738263 196 F196M M 1.647608 196 F196G G 1.590716196 F196S S 1.577837 196 F196Y Y 1.414589 196 F196V V 1.395387 196 F196II 1.320955 196 F196W W 1.014435

The following Table provides variants with PAF results that were betterthan those observed for wild-type M. smegmatis perhydrolase. In thisTable, the middle column indicates the amino acid residue in thewild-type perhydrolase (WT), followed by the position number and thevariant amino acid in that position (Var).

TABLE 10-2 Variants with PAF Values Better Than Wild-Type WT/Pos./Peracid formation Pos Var relative to WT 2 A002W 1.75 2 A002D 1.30 2A002F 1.24 2 A002I 1.18 2 A002G 1.15 2 A002S 1.01 3 K003Y 1.06 3 K003I1.05 3 K003L 1.04 3 K003T 1.01 3 K003H 1.01 4 R004Q 1.03 5 I005T 1.12 5I005S 1.02 6 L006V 1.07 6 L006I 1.07 6 L006T 1.06 7 C007K 2.69 7 C007Y2.09 7 C007I 1.76 7 C007H 1.73 7 C007A 1.42 7 C007G 1.39 7 C007M 1.13 8F008R 1.43 8 F008V 1.18 8 F008G 1.09 8 F008H 1.02 10 D010L 3.97 10 D010W3.18 10 D010K 2.13 10 D010Y 1.51 10 D010T 1.47 10 D010I 1.28 12 L012Q2.65 12 L012C 2.29 12 L012A 1.10 15 G015A 1.54 15 G015S 1.05 17 V017G1.17 17 V017R 1.10 17 V017A 1.01 18 P018Y 1.33 18 P018N 1.33 18 P018C1.26 18 P018E 1.22 18 P018V 1.19 18 P018R 1.16 18 P018Q 1.12 18 P018H1.12 18 P018G 1.07 19 V019G 1.32 19 V019S 1.24 19 V019R 1.03 19 V019L1.00 20 E020W 2.94 20 E020G 2.36 20 E020T 2.22 20 E020L 2.20 20 E020H2.17 20 E020V 2.11 20 E020S 2.01 20 E020C 1.57 20 E020N 1.40 20 E020A1.29 20 E020Q 1.27 21 D021K 1.58 21 D021W 1.55 21 D021L 1.46 21 D021A1.46 21 D021G 1.37 21 D021Y 1.30 21 D021F 1.30 21 D021S 1.24 22 G022A1.55 22 G022T 1.03 22 G022S 1.02 25 T025G 1.86 25 T025S 1.60 25 T025A1.33 25 T025I 1.02 26 E026M 2.00 26 E026A 1.93 26 E026R 1.48 26 E026K1.46 26 E026T 1.44 26 E026C 1.40 26 E026V 1.39 26 E026N 1.37 26 E026H1.33 26 E026L 1.30 26 E026G 1.28 26 E026S 1.27 26 E026W 1.25 27 R027K1.22 28 F028M 1.33 28 F028A 1.27 28 F028W 1.16 28 F028L 1.09 28 F028S1.05 29 A029W 1.91 29 A029V 1.80 29 A029R 1.76 29 A029Y 1.70 29 A029G1.60 29 A029S 1.49 29 A029T 1.42 29 A029E 1.12 29 A029C 1.08 30 P030K1.21 30 P030R 1.16 30 P030V 1.06 30 P030T 1.05 30 P030A 1.05 30 P030S1.03 30 P030Q 1.01 30 P030H 1.01 30 P030E 1.01 31 D031W 1.83 31 D031L1.81 31 D031T 1.45 31 D031G 1.44 31 D031F 1.44 31 D031N 1.34 31 D031V1.28 31 D031A 1.24 31 D031R 1.22 31 D031S 1.15 31 D031E 1.13 31 D031Q1.07 32 V032K 1.09 32 V032R 1.05 33 R033S 1.00 36 G036I 1.32 36 G036K1.27 36 G036L 1.24 37 V037S 1.40 37 V037I 1.26 37 V037A 1.25 37 V037H1.21 37 V037L 1.16 37 V037C 1.09 37 V037T 1.05 39 A039L 1.43 39 A039K1.36 39 A039Y 1.36 39 A039I 1.26 39 A039T 1.26 39 A039W 1.23 39 A039V1.21 39 A039G 1.17 39 A039R 1.17 39 A039E 1.09 40 Q040K 2.61 40 Q040I2.58 40 Q040W 2.39 40 Q040L 2.14 40 Q040T 2.01 40 Q040R 1.89 40 Q040Y1.83 40 Q040G 1.79 40 Q040S 1.57 40 Q040N 1.53 40 Q040D 1.16 40 Q040E1.08 41 Q041K 1.38 41 Q041R 1.19 41 Q041W 1.14 41 Q041H 1.12 41 Q041S1.11 41 Q041Y 1.09 41 Q041V 1.07 41 Q041A 1.03 41 Q041L 1.00 42 L042K2.46 42 L042W 2.06 42 L042H 1.92 42 L042R 1.38 42 L042G 1.17 42 L042T1.08 42 L042F 1.07 43 G043A 1.49 43 G043C 1.48 43 G043K 1.42 43 G043M1.37 43 G043Y 1.26 43 G043E 1.25 43 G043L 1.22 43 G043R 1.22 43 G043S1.18 43 G043H 1.17 43 G043P 1.08 44 A044F 2.84 44 A044V 2.13 44 A044C1.80 44 A044L 1.61 44 A044W 1.40 44 A044M 1.20 45 D045K 1.34 45 D045T1.27 45 D045R 1.16 45 D045W 1.15 45 D045S 1.13 45 D045G 1.13 45 D045H1.13 45 D045F 1.11 45 D045L 1.05 45 D045V 1.05 45 D045Q 1.04 45 D045A1.04 46 F046E 1.25 46 F046D 1.17 46 F046G 1.02 46 F046K 1.00 47 E047R2.45 47 E047T 1.96 47 E047P 1.36 47 E047S 1.28 47 E047H 1.27 47 E047G1.20 47 E047K 1.19 47 E047F 1.09 47 E047I 1.03 49 I049G 1.34 49 I049H1.27 49 I049S 1.24 49 I049K 1.23 49 I049V 1.20 49 I049L 1.14 49 I049Y1.07 49 I049R 1.05 49 I049E 1.02 49 I049M 1.01 50 E050L 1.19 50 E050M1.18 50 E050A 1.12 51 E051V 1.47 51 E051A 1.28 51 E051G 1.22 51 E051T1.18 51 E051L 1.11 51 E051I 1.07 53 L053H 5.05 53 L053Q 1.48 53 L053G1.32 53 L053S 1.16 53 L053T 1.02 54 S054P 5.20 54 S054I 4.78 54 S054V4.72 54 S054A 3.46 54 S054R 3.38 54 S054L 2.02 54 S054T 1.46 54 S054K1.44 54 S054G 1.43 54 S054C 1.26 54 S054Q 1.03 55 A055G 1.69 55 A055T1.69 57 T057S 1.63 57 T057R 1.61 57 T057V 1.28 57 T057I 1.19 59 N059W1.13 59 N059R 1.09 59 N059T 1.07 59 N059S 1.06 59 N059Q 1.02 60 I060H1.02 60 I060R 1.00 61 D061H 1.44 61 D061S 1.26 61 D061R 1.11 61 D061I1.08 61 D061F 1.01 62 D062E 1.02 63 P063G 1.71 63 P063T 1.50 63 P063M1.46 63 P063S 1.42 63 P063K 1.40 63 P063A 1.35 63 P063Y 1.35 63 P063W1.35 63 P063V 1.31 63 P063R 1.31 63 P063F 1.25 63 P063L 1.15 63 P063Q1.09 64 T064G 1.23 64 T064S 1.11 65 D065A 1.31 65 D065S 1.17 65 D065H1.10 66 P066R 1.85 66 P066V 1.83 66 P066H 1.59 66 P066I 1.59 66 P066G1.50 66 P066Q 1.46 66 P066T 1.41 66 P066S 1.39 66 P066Y 1.33 66 P066L1.14 66 P066N 1.12 67 R067N 1.58 67 R067G 1.39 67 R067T 1.28 67 R067F1.26 67 R067L 1.20 67 R067Q 1.16 67 R067W 1.07 67 R067E 1.04 67 R067P1.01 68 L068E 1.44 68 L068W 1.21 68 L068I 1.13 68 L068G 1.09 68 L068V1.09 68 L068H 1.05 68 L068T 1.03 69 N069V 1.99 69 N069K 1.72 69 N069R1.49 69 N069I 1.47 69 N069H 1.36 69 N069T 1.35 69 N069L 1.30 69 N069S1.21 69 N069G 1.20 69 N069Q 1.07 69 N069W 1.05 69 N069C 1.05 71 A071S1.75 71 A071T 1.70 71 A071H 1.70 71 A071G 1.59 71 A071I 1.51 71 A071E1.45 71 A071K 1.44 71 A071R 1.40 71 A071N 1.23 71 A071L 1.23 71 A071F1.13 71 A071C 1.01 72 S072L 1.26 72 S072H 1.21 72 S072G 1.20 72 S072T1.10 72 S072V 1.08 72 S072Y 1.07 73 Y073R 1.26 73 Y073Q 1.23 73 Y073S1.17 73 Y073K 1.07 74 L074S 2.72 74 L074G 1.95 74 L074W 1.38 75 P075R1.60 75 P075S 1.39 75 P075T 1.28 75 P075Q 1.21 75 P075G 1.16 75 P075H1.05 75 P075W 1.04 76 S076P 1.23 77 C077T 1.12 77 C077V 1.05 77 C077G1.01 78 L078G 4.98 78 L078H 4.82 78 L078E 3.01 78 L078N 2.68 78 L078T1.87 78 L078Q 1.73 78 L078V 1.53 78 L078I 1.43 78 L078Y 1.39 79 A079H1.93 79 A079L 1.80 79 A079I 1.59 79 A079M 1.50 79 A079N 1.48 79 A079Q1.47 79 A079R 1.47 79 A079W 1.27 79 A079T 1.17 79 A079E 1.12 80 T080C1.31 80 T080V 1.23 80 T080G 1.16 80 T080A 1.00 81 H081K 1.52 81 H081L1.23 81 H081N 1.17 81 H081G 1.17 81 H081A 1.15 81 H081C 1.13 81 H081W1.13 81 H081V 1.10 81 H081F 1.10 81 H081S 1.04 82 L082P 1.46 82 L082G1.38 82 L082R 1.34 82 L082H 1.33 82 L082K 1.19 82 L082T 1.18 82 L082I1.17 82 L082S 1.15 82 L082V 1.02 83 P083K 1.37 83 P083G 1.31 83 P083H1.27 83 P083R 1.19 83 P083S 1.17 84 L084K 1.10 84 L084H 1.01 85 D085Q3.09 85 D085R 2.38 85 D085S 2.28 85 D085H 1.55 85 D085N 1.54 85 D085G1.41 85 D085T 1.33 85 D085E 1.12 85 D085F 1.01 86 L086A 1.38 86 L086C1.16 86 L086G 1.15 88 I088H 1.20 88 I088T 1.03 88 I088G 1.01 90 M090T1.27 90 M090I 1.13 90 M090V 1.08 90 M090S 1.06 90 M090L 1.02 91 L091G1.21 91 L091T 1.06 92 G092V 1.49 92 G092S 1.26 93 T093Y 5.26 93 T093F3.52 93 T093A 1.38 93 T093C 1.08 95 D095E 2.04 96 T096S 1.04 97 K097R2.80 97 K097Q 1.14 98 A098L 2.22 98 A098H 2.09 98 A098I 2.05 98 A098Y2.02 98 A098S 1.73 98 A098T 1.72 98 A098G 1.57 98 A098C 1.30 98 A098N1.24 98 A098D 1.11 98 A098P 1.10 100 F100W 1.08 100 F100E 1.01 101 R101K1.24 103 T103W 1.26 103 T103Y 1.19 103 T103G 1.11 103 T103K 1.09 103T103I 1.08 103 T103L 1.05 104 P104H 2.84 104 P104T 2.70 104 P104G 2.67104 P104V 2.59 104 P104S 2.48 104 P104I 2.43 104 P104W 2.05 104 P104C1.95 104 P104E 1.84 104 P104F 1.79 104 P104N 1.62 104 P104R 1.62 104P104Q 1.34 104 P104M 1.09 105 L105P 1.71 105 L105C 1.56 105 L105F 1.30105 L105W 1.28 105 L105G 1.08 106 D106K 1.28 106 D106L 1.20 106 D106G1.18 106 D106H 1.09 106 D106E 1.08 106 D106T 1.06 106 D106I 1.04 106D106F 1.02 106 D106C 1.01 107 I107E 2.55 107 I107S 2.04 107 I107N 1.81107 I107G 1.76 107 I107V 1.00 108 A108L 1.41 108 A108T 1.05 109 L109N1.52 109 L109W 1.30 109 L109Q 1.18 109 L109Y 1.16 109 L109I 1.05 109L109D 1.00 111 M111K 1.98 111 M111I 1.95 111 M111L 1.55 111 M111T 1.49111 M111F 1.47 111 M111V 1.47 111 M111Y 1.43 111 M111S 1.03 112 S112L1.03 112 S112H 1.00 113 V113L 1.50 113 V113H 1.34 113 V113K 1.19 113V113R 1.13 113 V113Y 1.11 113 V113F 1.05 113 V113Q 1.03 115 V115W 1.23115 V115T 1.15 115 V115L 1.12 115 V115G 1.09 115 V115I 1.05 115 V115Y1.03 116 T116G 1.10 116 T116A 1.01 117 Q117H 2.33 117 Q117T 2.23 117Q117Y 2.23 117 Q117W 2.16 117 Q117V 2.15 117 Q117G 2.08 117 Q117A 2.05117 Q117S 1.95 117 Q117F 1.57 117 Q117R 1.56 117 Q117M 1.54 117 Q117E1.15 118 V118Y 1.25 118 V118K 1.13 118 V118G 1.08 120 T120S 1.09 121S121L 1.35 121 S121W 1.33 121 S121R 1.26 121 S121K 1.24 121 S121G 1.20121 S121C 1.18 121 S121N 1.14 121 S121T 1.13 121 S121A 1.12 121 S121V1.12 122 A122H 1.14 122 A122I 1.13 122 A122T 1.08 122 A122K 1.08 122A122V 1.04 122 A122S 1.03 123 G123D 1.73 123 G123V 1.40 123 G123P 1.32123 G123E 1.13 123 G123T 1.06 123 G123H 1.00 124 G124L 1.92 124 G124I1.85 124 G124T 1.64 124 G124H 1.59 124 G124V 1.44 124 G124F 1.32 124G124S 1.27 124 G124Y 1.23 124 G124R 1.14 124 G124Q 1.12 125 V125G 2.95125 V125S 1.94 125 V125A 1.69 125 V125P 1.50 125 V125R 1.30 125 V125D1.24 125 V125Y 1.08 125 V125I 1.01 126 G126T 1.58 126 G126P 1.17 126G126L 1.17 127 T127H 1.57 127 T127V 1.07 127 T127I 1.06 127 T127S 1.05128 T128L 1.06 128 T128K 1.06 130 P130T 1.19 130 P130H 1.17 130 P130K1.16 130 P130G 1.16 130 P130S 1.16 130 P130V 1.15 130 P130W 1.15 130P130I 1.12 130 P130L 1.12 130 P130R 1.11 130 P130F 1.08 130 P130E 1.00131 A131L 1.83 131 A131R 1.76 131 A131H 1.72 131 A131G 1.66 131 A131W1.61 131 A131V 1.59 131 A131P 1.52 131 A131Y 1.50 131 A131S 1.48 131A131E 1.36 131 A131D 1.31 131 A131Q 1.29 132 P132Y 1.57 132 P132S 1.13133 K133Y 1.12 133 K133L 1.05 133 K133H 1.02 134 V134G 1.71 134 V134T1.25 134 V134N 1.18 134 V134S 1.16 134 V134L 1.13 134 V134I 1.12 136V136T 1.13 137 V137M 1.22 137 V137L 1.09 137 V137T 1.08 137 V137A 1.07137 V137G 1.02 138 S138I 1.15 138 S138G 1.05 140 P140A 1.90 140 P140T1.74 140 P140S 1.31 141 P141L 2.32 141 P141I 2.29 141 P141H 2.07 141P141V 1.96 141 P141T 1.84 141 P141S 1.70 141 P141R 1.65 141 P141G 1.64141 P141Q 1.39 141 P141N 1.32 141 P141A 1.10 142 L142W 2.41 142 L142K1.60 142 L142F 1.05 143 A143K 3.16 143 A143H 2.90 143 A143L 2.51 143A143V 2.45 143 A143W 2.27 143 A143T 2.18 143 A143R 2.15 143 A143S 1.77143 A143Q 1.74 143 A143F 1.56 143 A143P 1.53 143 A143G 1.48 143 A143D1.45 143 A143E 1.43 143 A143C 1.39 143 A143N 1.30 144 P144Y 2.34 144P144K 2.09 144 P144H 1.94 144 P144F 1.82 144 P144R 1.76 144 P144S 1.69144 P144T 1.46 144 P144G 1.45 144 P144D 1.45 144 P144N 1.44 144 P144L1.43 144 P144Q 1.37 144 P144M 1.24 144 P144A 1.09 145 M145L 1.72 145M145F 1.49 145 M145R 1.15 145 M145W 1.15 145 M145C 1.02 145 M145T 1.01147 H147A 1.28 147 H147S 1.26 147 H147T 1.20 147 H147P 1.12 147 H147E1.11 148 P148V 2.43 148 P148K 1.79 148 P148L 1.64 148 P148A 1.64 148P148R 1.51 148 P148T 1.50 148 P148Y 1.46 148 P148S 1.46 148 P148E 1.42148 P148F 1.37 148 P148Q 1.33 148 P148D 1.03 150 F150L 1.29 150 F150E1.23 151 Q151D 1.47 151 Q151R 1.36 151 Q151P 1.35 151 Q151A 1.29 151Q151T 1.24 151 Q151M 1.24 151 Q151E 1.14 151 Q151K 1.07 151 Q151H 1.06151 Q151S 1.05 151 Q151C 1.05 151 Q151Y 1.01 152 L152V 1.22 152 L152K1.21 152 L152R 1.20 152 L152W 1.18 152 L152T 1.12 152 L152S 1.12 152L152Y 1.09 152 L152H 1.09 152 L152G 1.08 152 L152E 1.08 152 L152Q 1.07152 L152D 1.07 152 L152I 1.04 152 L152C 1.00 153 I153K 1.62 153 I153H1.46 153 I153T 1.27 153 I153L 1.27 153 I153F 1.23 153 I153A 1.19 154F154Y 1.32 155 E155T 1.49 155 E155R 1.47 155 E155L 1.31 155 E155Y 1.27155 E155K 1.23 155 E155G 1.17 155 E155S 1.08 155 E155D 1.08 155 E155F1.07 156 G156P 1.44 156 G156T 1.15 156 G156K 1.10 156 G156M 1.09 156G156C 1.07 156 G156N 1.07 156 G156R 1.05 156 G156H 1.04 156 G156S 1.02157 G157T 1.74 157 G157R 1.51 157 G157S 1.30 157 G157K 1.28 157 G157F1.27 157 G157V 1.23 157 G157H 1.14 157 G157I 1.11 158 E158H 2.40 158E158K 2.08 158 E158F 2.06 158 E158R 1.99 158 E158Y 1.77 158 E158W 1.77158 E158L 1.59 158 E158S 1.57 158 E158V 1.52 158 E158Q 1.49 158 E158C1.46 158 E158A 1.45 158 E158T 1.45 158 E158P 1.41 158 E158N 1.41 158E158M 1.39 158 E158I 1.38 158 E158D 1.35 159 Q159R 1.15 159 Q159C 1.13159 Q159S 1.10 159 Q159D 1.09 159 Q159A 1.08 159 Q159M 1.07 159 Q159P1.06 159 Q159L 1.02 161 T161R 3.61 161 T161Y 2.40 161 T161H 1.82 161T161W 1.41 161 T161I 1.40 161 T161V 1.27 161 T161L 1.25 161 T161Q 1.04162 T162K 1.22 162 T162R 1.17 162 T162W 1.15 162 T162Y 1.03 162 T162H1.02 163 E163L 1.50 163 E163Y 1.41 163 E163H 1.32 163 E163G 1.25 163E163W 1.21 163 E163V 1.13 163 E163R 1.12 163 E163S 1.12 163 E163A 1.11163 E163C 1.11 163 E163F 1.07 165 A165R 1.70 165 A165K 1.35 165 A165F1.23 165 A165Q 1.21 165 A165V 1.21 165 A165Y 1.20 165 A165T 1.18 165A165I 1.17 165 A165P 1.14 165 A165L 1.08 165 A165G 1.05 165 A165N 1.01165 A165S 1.00 166 R166Y 1.29 166 R166L 1.27 166 R166I 1.26 166 R166W1.25 166 R166H 1.20 166 R166T 1.19 166 R166V 1.17 166 R166K 1.17 166R166S 1.16 166 R166G 1.15 167 V167T 1.13 167 V167I 1.08 167 V167Y 1.07167 V167H 1.03 168 Y168G 1.89 168 Y168T 1.51 168 Y168V 1.19 169 S169Y1.26 169 S169R 1.24 169 S169K 1.21 169 S169I 1.16 169 S169T 1.15 169S169L 1.08 169 S169C 1.03 169 S169Q 1.02 170 A170K 1.71 170 A170G 1.59170 A170I 1.59 170 A170S 1.47 170 A170F 1.44 170 A170T 1.40 170 A170E1.28 170 A170D 1.27 170 A170N 1.21 170 A170V 1.20 170 A170C 1.15 170A170Q 1.15 170 A170L 1.05 170 A170W 1.04 170 A170M 1.03 171 L171K 2.05171 L171H 1.67 171 L171T 1.54 171 L171I 1.53 171 L171S 1.43 171 L171F1.30 171 L171G 1.26 171 L171Y 1.20 171 L171V 1.02 172 A172I 1.70 172A172S 1.59 172 A172W 1.43 172 A172G 1.41 172 A172V 1.40 172 A172T 1.25172 A172L 1.20 172 A172C 1.20 173 S173Y 1.19 173 S173K 1.17 173 S173W1.16 173 S173L 1.15 173 S173R 1.09 173 S173H 1.07 173 S173T 1.06 174F174G 1.60 174 F174P 1.54 174 F174Q 1.42 174 F174C 1.32 174 F174S 1.16174 F174L 1.05 175 M175T 2.21 175 M175G 2.04 175 M175V 1.93 175 M175L1.61 175 M175Q 1.56 175 M175R 1.55 175 M175N 1.39 175 M175W 1.25 176K176W 1.19 176 K176T 1.04 176 K176Y 1.04 176 K176V 1.04 176 K176G 1.01178 P178L 1.82 178 P178Y 1.38 178 P178K 1.34 178 P178W 1.14 178 P178G1.09 179 F179L 1.15 179 F179Y 1.05 180 F180L 1.30 180 F180I 1.20 180F180V 1.14 180 F180Y 1.12 180 F180W 1.11 180 F180K 1.08 180 F180T 1.01181 D181A 1.35 181 D181K 1.33 181 D181Y 1.29 181 D181W 1.26 181 D181L1.25 181 D181R 1.23 181 D181S 1.21 181 D181Q 1.14 181 D181E 1.10 181D181G 1.09 181 D181C 1.09 181 D181P 1.03 181 D181T 1.02 182 A182T 1.14184 S184Y 1.06 184 S184F 1.05 184 S184T 1.04 184 S184H 1.02 185 V185K1.37 185 V185Y 1.37 185 V185W 1.36 185 V185H 1.30 185 V185L 1.23 185V185R 1.15 185 V185G 1.12 185 V185T 1.11 185 V185S 1.09 185 V185I 1.07185 V185F 1.02 186 I186G 1.86 186 I186T 1.51 186 I186A 1.46 186 I186S1.39 186 I186V 1.28 186 I186L 1.17 186 I186F 1.01 187 S187K 1.45 187S187Y 1.43 187 S187I 1.38 187 S187L 1.37 187 S187W 1.30 187 S187H 1.29187 S187V 1.23 187 S187T 1.12 187 S187R 1.04 187 S187G 1.03 187 S187F1.02 188 T188Y 1.48 188 T188V 1.22 188 T188S 1.16 188 T188I 1.13 188T188H 1.11 188 T188R 1.01 189 D189L 1.30 189 D189H 1.25 189 D189W 1.09190 G190W 1.88 190 G190K 1.01 191 V191Y 1.32 191 V191H 1.30 191 V191W1.20 191 V191S 1.20 191 V191K 1.17 191 V191I 1.14 191 V191F 1.13 191V191R 1.05 191 V191L 1.04 196 F196H 1.77 196 F196L 1.77 196 F196C 1.74196 F196M 1.65 196 F196G 1.59 196 F196S 1.58 196 F196Y 1.41 196 F196V1.40 196 F196I 1.32 196 F196W 1.01 197 T197L 1.21 198 E198R 1.82 198E198I 1.80 198 E198V 1.60 198 E198W 1.59 198 E198L 1.57 198 E198P 1.52198 E198Y 1.48 198 E198C 1.38 198 E198F 1.37 198 E198Q 1.28 198 E198T1.25 198 E198N 1.24 198 E198M 1.18 198 E198S 1.06 199 A199C 1.77 199A199K 1.72 199 A199E 1.56 199 A199L 1.38 199 A199T 1.33 199 A199R 1.33199 A199V 1.32 199 A199D 1.31 199 A199H 1.27 199 A199Y 1.24 199 A199F1.23 199 A199S 1.20 199 A199G 1.14 199 A199M 1.07 201 N201Y 1.29 201N201F 1.16 201 N201G 1.08 202 R202W 1.97 202 R202F 1.89 202 R202E 1.69202 R202H 1.64 202 R202T 1.55 202 R202S 1.49 202 R202A 1.48 202 R202C1.44 202 R202M 1.43 202 R202L 1.43 202 R202G 1.39 202 R202I 1.33 203D203L 2.42 203 D203R 2.23 203 D203I 1.99 203 D203W 1.99 203 D203F 1.92203 D203H 1.84 203 D203C 1.78 203 D203S 1.66 203 D203V 1.66 203 D203G1.63 203 D203Q 1.60 203 D203A 1.53 203 D203E 1.34 203 D203N 1.05

The following Table, provides variants with a PAF PI greater than 1.5.

TABLE 10-3 PAF PI > 1.5 Wild-Type Residue/Pos. Variant Amino Acid(s) A2W C7 H, I, K, Y D10 K, L, W, Y L12 C, Q G15 A E20 C, G, H, L, S, T, V, WD21 K, W G22 A T25 G, S E26 A, M A29 G, R, V, W, Y D31 L, W Q40 G, I, K,L, N, R, S, T, W, Y L42 H, K, W A44 C, F, L, V E47 R, T L53 H S54 A, I,L, P, R, V A55 G, T T57 R, S P63 G P66 H, I, R, V R67 N N69 K, V A71 G,H, I, S, T L74 G, S P75 R L78 E, G, H, N, Q, T, V A79 H, I, L H81 K D85H, N, Q, R, S T93 F, Y D95 E K97 R A98 G, H, I, L, S, T, Y P104 C, E, F,G, H, I, N, R, S, T, V, W L105 C, P I107 E, G, N, S L109 N M111 I, K, LV113 L Q117 A, F, G, H, M, R, S, T, V, W, Y G123 D, H, I, L, T G124 I, LV125 A, G, P, S G126 T T127 H A131 G, H, L, P, R, V, W, Y P132 Y V134 GP140 A, T, P141 G, H, I, L, R, S, T, V L142 K, W A143 F, H, K, L, P, Q,R, S, T, V, W P144 F, H, K, R, S, Y M145 L P148 A, K, L, R, T, V I153 KG157 R, T E158 F, H, K, L, R, S, V, W, Y T161 H, R, Y A165 T Y168 G, TA170 G, I, K L171 H, I, K, T A172 I, S F174 G, P M175 G, L, Q, R, T, VP178 L F196 C, G, H, L, M, S G190 W E198 I, L, P, R, V, W A199 C, E, KR202 E, F, H, T, W D203 A, C, F, G, H, I, L, Q, R,S, V, W V206 E, F, G,H, K, R, S, A209 K E210 H, K, S, T, V, W Q211 K V212 W

Table 10-4 provides variants with PAF PI values greater than 2.0.

TABLE 10-4 Variants with PAF PI > 2.0 Wild-Type Residue/Pos. Amino AcidVariant(s) C7 K, Y D10 K. L, W L12 C, Q E20 G, H, L, S, T, V, W E26 MQ40 I, K, L, T, W L42 K, W A44 F, V E47 R L53 H S54 A, I, L, P, R, V L74S L78 E, G, H, N D85 Q, R, S T93 F, Y D95 E K97 R A98 H, I, L, Y P104 G,H, I, S, T. V, W I107 E, S Q117 A, G, H, T, V, W, Y V125 G P141 H, I, LL142 W A143 H, K, L, R, T, V, W P144 K, Y P148 V E158 F, H, K T161 R, YL171 K M175 G, T D203 L, R V206 E, F, K E210 T

The following Table provides PAD assay results for various variants.

TABLE 10-5 PAD Assay Results WT/Pos/ PAD Perf. Position Mutation VariantInd. 1 M001A A <0.01 1 M001E E <0.01 1 M001F F <0.01 1 M001G G <0.01 1M001K K <0.01 1 M001N N <0.01 1 M001P P <0.01 1 M001R R <0.01 1 M001S S<0.01 1 M001T T <0.01 1 M001W W <0.01 1 M001V V 0.944944 3 K003V V0.835476 4 R004L L <0.01 4 R004V V 0.079216 4 R004I I 0.153122 4 R004W W0.484006 4 R004G G 0.78952 4 R004S S 0.907174 4 R004E E 0.970668 4 R004YY 0.983327 4 R004H H 0.986096 4 R004Q Q 0.98766 4 R004T T 0.999841 5I005G G <0.01 5 I005N N <0.01 5 I005P P <0.01 5 I005R R <0.01 5 I005W W<0.01 5 I005F F 0.15045 5 I005S S 0.367738 5 I005H H 0.626022 5 I005T T0.7212 5 I005V V 0.917243 6 L006S S <0.01 6 L006K K <0.01 6 L006G G<0.01 6 L006H H <0.01 6 L006R R <0.01 6 L006W W <0.01 6 L006E E <0.01 6L006Q Q <0.01 6 L006V V 0.352616 6 L006T T 0.354148 6 L006I I 0.819654 7C007S S <0.01 7 C007R R <0.01 7 C007L L <0.01 7 C007P P <0.01 7 C007T T<0.01 7 C007W W <0.01 7 C007Y Y 0.544454 7 C007M M 0.678238 7 C007G G0.686018 10 D010W W <0.01 10 D010K K <0.01 10 D010Y Y <0.01 10 D010T T<0.01 10 D010I I <0.01 10 D010V V <0.01 10 D010S S <0.01 10 D010G G<0.01 10 D010R R <0.01 10 D010A A <0.01 10 D010M M <0.01 10 D010N N<0.01 10 D010P P <0.01 10 D010E E 0.147899 11 S011T T <0.01 11 S011V V<0.01 11 S011D D <0.01 11 S011E E <0.01 11 S011F F <0.01 11 S011G G<0.01 11 S011L L <0.01 11 S011Q Q <0.01 11 S011R R <0.01 11 S011H H0.332012 11 S011K K 0.399168 11 S011A A 0.528328 11 S011I I 0.562735 12L012V V <0.01 12 L012S S <0.01 12 L012G G <0.01 12 L012R R <0.01 12L012D D <0.01 12 L012P P <0.01 12 L012W W <0.0162738575856614 12 L012T T0.064264 12 L012A A 0.074567 12 L012K K 0.134919 12 L012H H 0.164894 12L012F F 0.171369 12 L012Q Q 0.219754 12 L012C C 0.221492 12 L012N N0.655242 13 T013F F <0.01 13 T013R R <0.01 13 T013W W <0.01 13 T013Q Q0.508867 13 T013V V 0.625148 13 T013S S 0.682494 13 T013G G 0.768701 14W014I I <0.01 14 W014S S <0.01 14 W014G G <0.01 14 W014K K <0.01 14W014V V <0.01 14 W014L L <0.01 14 W014T T <0.01 14 W014R R <0.01 14W014N N <0.01 14 W014P P <0.01 14 W014E E 0.150043 14 W014F F 0.21807314 W014A A 0.271277 14 W014Y Y 0.64896 14 W014W W 0.989643 15 G015C C<0.01 15 G015N N <0.01 15 G015D D <0.01 15 G015E E <0.01 15 G015H H<0.01 15 G015K K <0.01 15 G015L L <0.01 15 G015P P <0.01 15 G015R R<0.01 15 G015Y Y <0.01 15 G015A A 0.614319 15 G015S S 0.631317 16 W016SS <0.01 16 W016G G <0.01 16 W016H H <0.01 16 W016N N <0.01 16 W016R R<0.01 16 W016T T <0.01 16 W016P P 0.150383 16 W016Q Q 0.312038 16 W016MM 0.370155 16 W016A A 0.553088 16 W016D D 0.569713 16 W016E E 0.64737516 W016V V 0.875327 17 V017A A 0.675391 17 V017E E 0.749717 17 V017G G0.838345 17 V017K K 0.844479 17 V017F F 0.847091 17 V017T T 0.861827 17V017Y Y 0.876678 17 V017R R 0.936013 17 V017P P 0.956795 17 V017I I0.993337 17 V017L L 0.996217 18 P018A A <0.01 18 P018M M <0.01 18 P018SS 0.066689 19 V019P P <0.01 19 V019M M 0.117174 19 V019R R 0.343385 19V019Q Q 0.395965 19 V019A A 0.554598 19 V019G G 0.55596 19 V019S S0.573928 19 V019E E 0.620236 19 V019Y Y 0.696626 19 V019D D 0.785756 19V019L L 0.910961 19 V019K K 0.965611 21 D021V V <0.01 21 D021P P0.534939 21 D021S S 0.689672 21 D021E E 0.864655 21 D021F F 0.876655 21D021W W 0.894205 21 D021L L 0.971454 22 G022K K <0.01 22 G022W W0.231005 22 G022R R 0.563069 22 G022V V 0.850851 22 G022S S 0.981692 23A023R R 0.283095 23 A023S S 0.335177 23 A023G G 0.350575 23 A023F F0.438047 23 A023V V 0.598414 23 A023Q Q 0.732052 23 A023P P 0.733451 23A023W W 0.801206 23 A023M M 0.946802 23 A023Y Y 0.962455 24 P024S S0.614708 24 P024Q Q 0.652848 24 P024T T 0.663925 24 P024A A 0.681992 24P024G G 0.755229 24 P024I I 0.853247 24 P024R R 0.907892 24 P024H H0.969695 25 T025P P <0.01 25 T025H H <0.01 25 T025L L <0.01 25 T025R R<0.01 25 T025M M <0.01 25 T025E E <0.01 25 T025D D <0.01 25 T025K K0.133406 25 T025W W 0.144315 25 T025I I 0.350917 25 T025G G 0.426214 25T025C C 0.509792 25 T025V V 0.514769 25 T025S S 0.576256 25 T025A A0.863346 26 E026S S 0.280953 26 E026T T 0.39705 26 E026W W 0.471182 26E026N N 0.47572 26 E026R R 0.813632 26 E026G G 0.869755 26 E026C C0.939981 26 E026V V 0.966156 26 E026P P 0.993535 27 R027W W <0.01 27R027T T <0.0149789677895526 27 R027P P 0.483512 27 R027C C 0.58498 27R027S S 0.686775 27 R027G G 0.836174 27 R027E E 0.925988 27 R027V V0.943209 28 F028G G <0.01 28 F028H H <0.01 28 F028I I <0.01 28 F028R R<0.01 28 F028P P 0.385272 28 F028V V 0.531941 28 F028S S 0.696363 29A029V V 0.43718 29 A029T T 0.467508 29 A029S S 0.546873 29 A029Y Y0.593264 29 A029P P 0.622623 29 A029R R 0.728312 29 A029W W 0.738583 29A029M M 0.768108 29 A029G G 0.802278 29 A029E E 0.844095 29 A029D D0.996225 30 P030M M 0.78893 30 P030Q Q 0.905135 30 P030A A 0.918048 31D031E E 0.882779 32 V032P P <0.01 32 V032R R 0.715259 33 R033D D <0.0133 R033E E <0.01 33 R033H H <0.01 33 R033P P <0.01 33 R033W W <0.01 33R033V V 0.935183 34 W034R R <0.01 34 W034E E <0.01 34 W034K K <0.01 34W034Q Q 0.041311 34 W034S S 0.079486 34 W034T T 0.153641 34 W034V V0.72591 34 W034G G 0.880049 34 W034I I 0.93831 35 T035Q Q <0.01 35 T035NN <0.01 35 T035R R <0.01 35 T035K K <0.01 35 T035L L <0.01 35 T035P P<0.01 35 T035W W <0.01 35 T035Y Y <0.01 35 T035V V 0.344374 36 G036P P<0.01 36 G036S S 0.25722 36 G036T T 0.326076 36 G036V V 0.375828 36G036M M 0.536338 36 G036N N 0.557724 36 G036W W 0.682701 36 G036Q Q0.712029 36 G036R R 0.897684 38 L038K K <0.01 38 L038G G <0.01 38 L038EE <0.01 38 L038P P <0.01 38 L038Q Q <0.01 38 L038R R <0.01 38 L038W W<0.01 40 Q040P P <0.01 41 Q041V V <0.01 41 Q041S S 0.222419 41 Q041P P0.662368 41 Q041Y Y 0.701492 41 Q041W W 0.878483 42 L042W W <0.01 42L042H H <0.01 42 L042T T <0.01 42 L042D D <0.01 42 L042Q Q 0.280991 42L042S S 0.450557 42 L042R R 0.64188 42 L042I I 0.658658 42 L042V V0.725221 42 L042M M 0.73687 42 L042G G 0.759964 43 G043S S 0.233902 43G043P P 0.310899 43 G043V V 0.332639 43 G043Q Q 0.475759 43 G043R R0.585481 43 G043C C 0.725373 43 G043I I 0.766408 43 G043K K 0.856798 43G043M M 0.877674 43 G043Y Y 0.944457 43 G043H H 0.957156 44 A044S S<0.01 44 A044Y Y <0.01 44 A044T T <0.01 44 A044R R <0.01 44 A044D D<0.01 44 A044H H <0.01 44 A044P P <0.01 44 A044E E 0.028463 44 A044V V0.504951 44 A044F F 0.803847 44 A044W W 0.847767 44 A044M M 0.975188 44A044L L 0.99381 45 D045S S 0.382964 45 D045T T 0.438291 45 D045R R0.492492 45 D045V V 0.500129 45 D045P P 0.531241 45 D045Q Q 0.568687 45D045W W 0.582004 45 D045H H 0.779564 45 D045L L 0.781626 45 D045M M0.78286 45 D045G G 0.839279 45 D045A A 0.841569 45 D045C C 0.844725 45D045K K 0.867296 46 F046H H <0.01 46 F046T T 0.429962 46 F046W W0.633171 46 F046S S 0.656356 46 F046V V 0.786355 46 F046I I 0.882982 46F046G G 0.944614 47 E047P P 0.357072 47 E047R R 0.620501 47 E047N N0.627512 47 E047S S 0.628088 47 E047M M 0.703134 47 E047A A 0.757492 47E047F F 0.763159 47 E047C C 0.772744 47 E047T T 0.837562 47 E047D D0.975388 47 E047H H 0.99217 48 V048R R <0.01 48 V048W W <0.01 48 V048S S0.423613 48 V048G G 0.873544 48 V048N N 0.980906 48 V048E E 0.987222 49I049P P 0.161279 49 I049R R 0.29139 49 I049W W 0.676641 49 I049H H0.740799 49 I049S S 0.789362 49 I049E E 0.876247 49 I049V V 0.972022 50E050R R <0.01 50 E050W W 0.14091 50 E050V V 0.425221 50 E050I I 0.57536950 E050S S 0.645021 50 E050Q Q 0.906441 50 E050L L 0.967983 51 E051R R<0.01 51 E051P P <0.01 51 E051I I 0.044391 51 E051W W 0.165053 51 E051VV 0.367755 51 E051Q Q 0.761883 51 E051L L 0.927544 52 G052H H <0.01 52G052S S <0.01 52 G052V V <0.01 52 G052T T <0.01 52 G052M M <0.01 52G052F F <0.01 52 G052I I 0.069022 52 G052P P 0.242545 52 G052L L0.244397 52 G052Q Q 0.283827 52 G052R R 0.349923 52 G052E E 0.549067 52G052A A 0.793929 53 L053R R <0.01 53 L053W W <0.01 53 L053P P <0.01 53L053D D <0.01328259968325 53 L053E E 0.191623 53 L053K K 0.237686 53L053S S 0.260431 53 L053G G 0.32712 53 L053V V 0.652864 53 L053I I0.659806 53 L053Q Q 0.717093 53 L053T T 0.842042 54 S054F F <0.01 54S054W W <0.01 54 S054H H <0.01 54 S054K K 0.083519 54 S054I I 0.11629554 S054Y Y 0.124722 54 S054G G 0.170484 54 S054L L 0.258821 54 S054V V0.285755 54 S054E E 0.296919 54 S054T T 0.329279 54 S054R R 0.354857 54S054M M 0.482666 54 S054Q Q 0.531633 54 S054D D 0.647787 54 S054C C0.87772 55 A055V V <0.01 55 A055I I <0.01 55 A055P P <0.01 55 A055W W<0.01 55 A055Y Y 0.176777 55 A055R R 0.245648 55 A055T T 0.415054 55A055G G 0.731513 55 A055L L 0.866592 55 A055S S 0.866756 55 A055H H0.921909 56 R056C C <0.01 56 R056G G <0.01 56 R056T T <0.01 56 R056E E<0.01 56 R056H H <0.01 56 R056K K <0.01 56 R056P P <0.01 56 R056Q Q<0.01 56 R056W W <0.01 56 R056Y Y <0.01 56 R056S S 0.123501 56 R056L L0.237933 56 R056N N 0.267811 56 R056A A 0.68802 57 T057R R <0.01 57T057P P <0.01 57 T057W W <0.01 57 T057N N 0.245605 57 T057C C 0.39800157 T057Y Y 0.551709 57 T057H H 0.605386 57 T057A A 0.651879 57 T057L L0.762087 57 T057V V 0.86913 57 T057I I 0.870692 58 T058E E <0.01 58T058G G <0.01 58 T058K K <0.01 58 T058P P <0.01 58 T058R R <0.01 58T058W W <0.01 58 T058Y Y <0.01 58 T058M M 0.026886 58 T058A A 0.36125858 T058V V 0.955494 58 T058S S 0.964758 59 N059R R <0.01 59 N059M M<0.01 59 N059P P <0.01 59 N059Q Q 0.165409 59 N059T T 0.501362 59 N059SS 0.651989 59 N059K K 0.731191 59 N059E E 0.879272 59 N059V V 0.88734159 N059G G 0.890006 59 N059F F 0.911279 59 N059A A 0.929578 59 N059Y Y0.99189 59 N059C C 0.99959 60 I060P P 0.318965 60 I060D D 0.660273 60I060C C 0.668516 60 I060M M 0.682237 60 I060A A 0.788799 60 I060R R0.809655 60 I060L L 0.913226 60 I060E E 0.923286 60 I060K K 0.959958 60I060S S 0.999829 61 D061F F 0.698154 61 D061A A 0.708121 61 D061C C0.848446 61 D061Y Y 0.948278 61 D061V V 0.968066 61 D061N N 0.999276 62D062T T <0.01 62 D062I I <0.01 62 D062V V <0.01 62 D062H H <0.01 62D062W W <0.01 62 D062S S <0.01 62 D062L L <0.01 62 D062G G <0.01 62D062R R <0.01 62 D062M M <0.01 62 D062P P <0.01 62 D062Q Q <0.01 62D062A A 0.113753 62 D062C C 0.490736 62 D062E E 0.602369 63 P063A A0.598416 63 P063R R 0.801911 63 P063S S 0.898408 63 P063M M 0.908904 63P063F F 0.925844 63 P063Y Y 0.948378 64 T064R R 0.106209 64 T064D D0.640095 64 T064W W 0.691185 64 T064Q Q 0.865168 64 T064C C 0.876862 64T064P P 0.936023 64 T064H H 0.960718 64 T064N N 0.983933 64 T064S S0.987972 65 D065V V 0.199467 65 D065R R 0.215599 65 D065H H 0.398178 65D065Y Y 0.42301 65 D065P P 0.423122 65 D065S S 0.468174 65 D065W W0.50219 65 D065T T 0.5039 65 D065G G 0.51655 65 D065I I 0.617391 65D065A A 0.723321 66 P066N N 0.381273 66 P066Q Q 0.422614 66 P066G G0.444859 66 P066R R 0.508806 66 P066C C 0.523524 66 P066A A 0.563865 66P066F F 0.672865 66 P066Y Y 0.699931 66 P066D D 0.718749 66 P066I I0.844376 66 P066V V 0.89302 66 P066H H 0.947771 66 P066L L 0.987271 67R067F F <0.0149736260903786 67 R067W W <0.0171329732205367 67 R067P P0.036575 67 R067E E 0.113415 67 R067V V 0.1203 67 R067Q Q 0.126838 67R067L L 0.156654 67 R067A A 0.215271 67 R067T T 0.315404 67 R067N N0.333066 67 R067G G 0.40823 67 R067K K 0.986487 68 L068G G <0.01 68L068A A <0.01 68 L068M M 0.02834 68 L068C C 0.05996 68 L068S S 0.07162268 L068N N 0.100981 68 L068E E 0.131505 68 L068H H 0.222734 68 L068Q Q0.254448 68 L068F F 0.254797 68 L068T T 0.324904 68 L068P P 0.35297 68L068D D 0.443469 68 L068Y Y 0.447862 68 L068R R 0.465293 68 L068V V0.507389 68 L068W W 0.561612 68 L068I I 0.727312 69 N069Y Y 0.173925 69N069W W 0.55063 69 N069P P 0.591783 69 N069R R 0.828172 69 N069G G0.976332 70 G070M M <0.01 70 G070T T <0.01 70 G070P P <0.01 70 G070V V<0.01 70 G070C C <0.01 70 G070R R <0.01 70 G070Y Y <0.01 70 G070K K<0.01 70 G070N N <0.01 70 G070Q Q <0.01 70 G070F F <0.01 70 G070I I0.270463 70 G070E E 0.33356 70 G070S S 0.638917 71 A071P P <0.01 71A071N N 0.613838 71 A071D D 0.646588 71 A071G G 0.675895 71 A071S S0.693249 71 A071R R 0.771492 71 A071H H 0.781953 71 A071I I 0.786894 71A071T T 0.79386 71 A071E E 0.809505 71 A071L L 0.838126 71 A071F F0.985677 71 A071C C 0.993683 72 S072Y Y 0.069096 72 S072W W 0.339835 72S072P P 0.555612 72 S072Q Q 0.655328 72 S072L L 0.703483 72 S072R R0.742354 72 S072D D 0.800127 72 S072V V 0.82827 72 S072E E 0.930527 72S072T T 0.973836 73 Y073P P <0.01 73 Y073R R 0.262561 73 Y073L L0.497588 73 Y073G G 0.509699 73 Y073H H 0.515737 73 Y073I I 0.641914 73Y073S S 0.676285 73 Y073V V 0.73535 73 Y073N N 0.758401 73 Y073D D0.803442 73 Y073Q Q 0.866092 73 Y073K K 0.944166 76 S076W W <0.01 76S076Y Y 0.177113 76 S076F F 0.461095 76 S076Q Q 0.900789 77 C077Y Y<0.01 77 C077R R <0.01 77 C077W W <0.01 77 C077F F <0.01 77 C077N N<0.01 77 C077P P <0.01 77 C077G G 0.181068 77 C077L L 0.734708 77 C077SS 0.764136 77 C077V V 0.802259 77 C077A A 0.912937 78 L078E E <0.01 78L078N N <0.01 78 L078A A <0.01 78 L078P P <0.01 78 L078R R <0.01 78L078S S <0.01 78 L078M M 0.477538 78 L078Q Q 0.519566 78 L078C C0.779536 78 L078Y Y 0.809511 78 L078V V 0.827484 79 A079H H <0.01 79A079F F <0.01 79 A079V V <0.01 79 A079C C 0.026887 79 A079Q Q 0.26870479 A079E E 0.272158 79 A079N N 0.281684 79 A079M M 0.284387 79 A079R R0.321618 79 A079W W 0.530746 79 A079T T 0.598368 79 A079I I 0.673986 79A079S S 0.779628 79 A079G G 0.915372 79 A079P P 0.94147 79 A079L L0.958677 80 T080W W <0.01 80 T080L L <0.01 80 T080K K <0.01 80 T080R R<0.01 80 T080E E <0.01 80 T080P P <0.01 80 T080H H 0.049717 80 T080Y Y0.107973 80 T080I I 0.146188 80 T080N N 0.529867 82 L082R R <0.01 82L082S S <0.01 82 L082W W <0.01 82 L082V V 0.187819 82 L082G G 0.31082382 L082T T 0.377413 82 L082H H 0.468806 82 L082I I 0.508005 82 L082K K0.508537 82 L082P P 0.516154 82 L082A A 0.976228 83 P083T T <0.01 83P083V V 0.186837 83 P083L L 0.211018 83 P083H H 0.611439 83 P083W W0.621496 83 P083G G 0.677444 83 P083S S 0.789585 83 P083Q Q 0.818267 83P083D D 0.831344 83 P083F F 0.99445 84 L084W W <0.01 84 L084V V 0.41657684 L084P P 0.43025 84 L084T T 0.438956 84 L084A A 0.453182 84 L084Q Q0.516002 84 L084S S 0.550862 84 L084R R 0.565943 84 L084N N 0.665228 84L084K K 0.79008 84 L084D D 0.85276 84 L084I I 0.870124 84 L084H H0.993217 85 D085I I 0.100248 85 D085L L 0.241561 85 D085V V 0.25268 85D085W W 0.341677 85 D085P P 0.543807 85 D085Y Y 0.554364 85 D085S S0.675803 85 D085T T 0.708548 85 D085N N 0.781957 85 D085Q Q 0.988545 86L086H H <0.01 86 L086S S <0.01 86 L086R R <0.01 86 L086E E <0.01 86L086F F <0.01 86 L086Q Q <0.01 86 L086W W 0.077717 86 L086V V 0.12013386 L086T T 0.284184 86 L086G G 0.696393 86 L086Y Y 0.815121 86 L086P P0.987233 87 V087S S <0.01 87 V087G G <0.01 87 V087Y Y <0.01 87 V087R R<0.01 87 V087K K <0.01 87 V087D D <0.01 87 V087F F 0.103908 87 V087T T0.147618 87 V087A A 0.16806 87 V087M M 0.751854 89 I089H H <0.01 89I089S S <0.01 89 I089G G <0.01 89 I089W W <0.01 89 I089Q Q <0.01 89I089D D <0.01 89 I089E E <0.01 89 I089R R <0.01 89 I089F F 0.745747 89I089V V 0.820031 89 I089T T 0.900425 94 N094L L <0.01 94 N094T T <0.0194 N094V V <0.01 94 N094H H <0.01 94 N094R R <0.01 94 N094W W <0.01 94N094M M 0.031458 94 N094C C 0.072751 94 N094Y Y 0.123924 94 N094G G0.532837 94 N094A A 0.74316 94 N094P P 0.789771 94 N094S S 0.877698 95D095A A <0.01 95 D095C C <0.01 95 D095G G <0.01 95 D095H H <0.01 95D095K K <0.01 95 D095L L <0.01 95 D095N N <0.01 95 D095Q Q <0.01 95D095R R <0.01 95 D095S S <0.01 95 D095T T <0.01 95 D095V V <0.01 95D095W W <0.01 95 D095Y Y <0.01 95 D095E E 0.754335 96 T096I I <0.01 96T096W W <0.01 96 T096Y Y <0.01 96 T096R R 0.136108 96 T096V V 0.58611 96T096S S 0.786547 96 T096P P 0.885134 97 K097Q Q <0.01 97 K097G G <0.0197 K097I I <0.01 97 K097W W <0.01 97 K097L L <0.01 97 K097V V <0.01 97K097Y Y <0.01 97 K097S S <0.01 97 K097T T <0.01 97 K097D D <0.01 97K097M M 0.216645 97 K097A A 0.227977 97 K097P P 0.26585 97 K097R R0.587184 99 Y099R R 0.291941 99 Y099V V 0.311502 99 Y099S S 0.367181 99Y099W W 0.566038 99 Y099H H 0.591623 99 Y099I I 0.60574 99 Y099G G0.700083 99 Y099P P 0.813989 99 Y099A A 0.822549 99 Y099L L 0.856204 100F100W W <0.01 100 F100K K <0.01 100 F100D D <0.01 100 F100E E 0.152427100 F100S S 0.852784 101 R101W W <0.01 101 R101K K 0.068708 101 R101Q Q0.107171 101 R101V V 0.442582 101 R101D D 0.800722 101 R101Y Y 0.803109101 R101P P 0.855496 101 R101N N 0.918012 101 R101C C 0.946306 101 R101II 0.955711 101 R101F F 0.965422 102 R102W W <0.01 102 R102F F 0.226881102 R102G G 0.270733 102 R102C C 0.363718 102 R102V V 0.60605 102 R102DD 0.684234 102 R102P P 0.894709 102 R102S S 0.960127 103 T103W W <0.01103 T103Y Y <0.01 103 T103G G <0.01 103 T103K K <0.01 103 T103I I <0.01103 T103L L <0.01 103 T103H H <0.01 103 T103A A <0.01 103 T103V V <0.01103 T103S S <0.01 103 T103C C <0.01 103 T103R R <0.01 103 T103N N <0.01103 T103F F <0.01 103 T103P P <0.01 104 P104R R <0.01 104 P104A A <0.01104 P104L L <0.01 104 P104W W 0.232802 104 P104T T 0.333526 104 P104S S0.529113 104 P104Q Q 0.847699 104 P104F F 0.863543 104 P104G G 0.984538105 L105V V <0.01 105 L105A A <0.01 105 L105M M <0.01 105 L105E E0.528458 105 L105S S 0.609931 105 L105Y Y 0.620029 105 L105T T 0.638962105 L105P P 0.902642 106 D106R R 0.559786 106 D106Q Q 0.617485 106 D106PP 0.632087 106 D106N N 0.642667 106 D106M M 0.855673 106 D106I I0.915931 106 D106L L 0.99561 107 I107E E <0.01 107 I107G G <0.01 107I107F F <0.01 107 I107Q Q <0.01 107 I107R R <0.01 107 I107H H <0.01 107I107W W <0.01 107 I107P P 0.318743 107 I107Y Y 0.524182 107 I107A A0.795478 107 I107N N 0.929935 107 I107V V 0.96863 108 A108D D <0.01 108A108F F <0.01 108 A108H H <0.01 108 A108I I <0.01 108 A108N N <0.01 108A108P P <0.01 108 A108R R <0.01 108 A108E E 0.60726 108 A108Q Q 0.734472108 A108T T 0.865471 108 A108V V 0.950481 109 L109W W <0.01 109 L109D D0.106206 109 L109I I 0.144257 109 L109E E 0.194168 109 L109R R 0.210346109 L109H H 0.220153 109 L109Q Q 0.222755 109 L109F F 0.317718 109 L109AA 0.323528 109 L109S S 0.378623 109 L109P P 0.434661 109 L109G G 0.51022109 L109V V 0.539733 109 L109M M 0.628881 109 L109N N 0.658369 109 L109TT 0.79132 109 L109Y Y 0.825105 110 G110T T <0.01 110 G110L L <0.01 110G110W W <0.01 110 G110Y Y <0.01 110 G110P P 0.224284 110 G110I I0.232219 110 G110S S 0.30218 110 G110Q Q 0.343918 110 G110R R 0.476072110 G110H H 0.73456 110 G110N N 0.770851 110 G110M M 0.816422 111 M111RR <0.01 111 M111S S 0.139078 111 M111H H 0.192733 111 M111G G 0.315165111 M111P P 0.566892 111 M111E E 0.668985 111 M111L L 0.67115 111 M111KK 0.706165 111 M111T T 0.763332 111 M111F F 0.776934 111 M111D D 0.78777111 M111V V 0.92522 112 S112Y Y <0.01 112 S112R R <0.01 112 S112P P<0.01 112 S112H H 0.380254 112 S112V V 0.479716 112 S112M M 0.564157 112S112W W 0.582165 112 S112K K 0.678369 112 S112T T 0.721644 112 S112N N0.850159 112 S112F F 0.878895 112 S112A A 0.943049 113 V113S S 0.572415113 V113G G 0.579385 113 V113K K 0.716865 113 V113H H 0.763416 113 V113WW 0.803685 113 V113L L 0.854963 113 V113T T 0.861744 113 V113D D0.871104 113 V113E E 0.936465 113 V113C C 0.937598 113 V113F F 0.959822113 V113Y Y 0.981976 114 L114H H <0.01 114 L114E E <0.01 114 L114F F<0.01 114 L114K K <0.01 114 L114R R <0.01 114 L114W W <0.01 114 L114Y Y<0.01 114 L114Q Q 0.115737 114 L114P P 0.275464 114 L114S S 0.545726 114L114V V 0.595416 114 L114N N 0.77333 115 V115H H <0.01 115 V115K K <0.01115 V115I I 0.994833 116 T116Y Y 0.466112 116 T116V V 0.571817 116 T116RR 0.619823 116 T116L L 0.681201 116 T116W W 0.748358 116 T116I I0.760474 116 T116Q Q 0.768867 116 T116P P 0.836786 116 T116G G 0.901886116 T116E E 0.906124 116 T116A A 0.952003 116 T116S S 0.963005 117 Q117WW 0.707035 117 Q117V V 0.761971 117 Q117G G 0.794858 117 Q117S S 0.86512118 V118K K <0.01 118 V118W W <0.01 118 V118E E <0.01 118 V118R R0.069623 118 V118P P 0.222399 118 V118D D 0.40168 118 V118I I 0.545694118 V118G G 0.559239 118 V118S S 0.815888 118 V118A A 0.852723 118 V118TT 0.91759 118 V118M M 0.933469 118 V118F F 0.998467 119 L119G G <0.01119 L119S S <0.01 119 L119F F <0.01 119 L119R R <0.01 119 L119P P <0.01119 L119T T 0.102922 119 L119N N 0.113151 119 L119V V 0.150373 119 L119WW 0.203313 119 L119C C 0.244106 119 L119D D 0.280381 119 L119E E0.322167 119 L119I I 0.427476 119 L119H H 0.462912 119 L119Y Y 0.556343120 T120P P <0.01 120 T120H H 0.498304 120 T120R R 0.599376 120 T120A A0.663543 120 T120Q Q 0.781096 120 T120C C 0.924433 121 S121P P 0.384623121 S121R R 0.701237 121 S121W W 0.772781 121 S121K K 0.77795 121 S121GG 0.992545 122 A122G G <0.01 122 A122D D 0.059137 122 A122F F 0.148369122 A122H H 0.169443 122 A122R R 0.396041 122 A122S S 0.431258 122 A122KK 0.450105 122 A122E E 0.467766 122 A122T T 0.520454 122 A122P P0.548155 122 A122I I 0.647406 122 A122N N 0.704284 122 A122Q Q 0.741587122 A122W W 0.862265 122 A122V V 0.886387 122 A122M M 0.938855 124 G124II <0.01 124 G124H H <0.01 124 G124M M <0.01 124 G124W W <0.01 124 G124PP <0.01 124 G124A A 0.031196 124 G124Q Q 0.208313 124 G124T T 0.315233124 G124V V 0.329769 124 G124R R 0.409769 124 G124L L 0.536625 124 G124SS 0.555215 124 G124Y Y 0.559199 124 G124N N 0.599171 124 G124D D 0.63784124 G124C C 0.672179 124 G124F F 0.950801 125 V125W W 0.24527 125 V125EE 0.385171 125 V125R R 0.466062 125 V125C C 0.541228 125 V125D D0.541318 125 V125P P 0.622352 125 V125F F 0.627367 125 V125S S 0.790998125 V125Y Y 0.813593 125 V125A A 0.925641 125 V125I I 0.941326 126 G126II <0.010426347441542 126 G126V V 0.175001 126 G126Y Y 0.234673 126 G126LL 0.540613 126 G126A A 0.552538 126 G126E E 0.599533 126 G126P P0.673809 126 G126T T 0.737666 126 G126R R 0.761417 126 G126N N 0.846727126 G126S S 0.902662 126 G126C C 0.980807 127 T127L L <0.01 127 T127E E<0.01 127 T127Q Q 0.151533 127 T127I I 0.203586 127 T127H H 0.60105 127T127D D 0.61747 127 T127M M 0.639504 127 T127C C 0.653314 127 T127V V0.683337 127 T127G G 0.710564 127 T127P P 0.773291 127 T127S S 0.828003128 T128D D 0.662836 129 Y129W W <0.01 129 Y129G G <0.01 129 Y129K K<0.01 129 Y129V V <0.01 129 Y129T T 0.138769 129 Y129A A 0.173554 129Y129R R 0.178362 129 Y129M M 0.211662 129 Y129D D 0.228506 129 Y129L L0.270643 129 Y129N N 0.530034 129 Y129P P 0.588917 129 Y129C C 0.610384129 Y129S S 0.692051 129 Y129F F 0.713199 146 P146W W 0.680806 146 P146TT 0.756105 146 P146V V 0.768041 146 P146S S 0.956673 148 P148Q Q0.975963 149 W149R R <0.01 149 W149E E <0.01 149 W149P P <0.01 149 W149CC 0.1164 149 W149I I 0.235936 149 W149A A 0.311848 149 W149S S 0.329233149 W149Q Q 0.402387 149 W149T T 0.440303 149 W149G G 0.44856 149 W149MM 0.494615 149 W149F F 0.495779 149 W149L L 0.637667 149 W149Y Y0.747652 150 F150P P 0.31768 150 F150N N 0.362798 150 F150G G 0.458431150 F150V V 0.511676 150 F150A A 0.539571 150 F150T T 0.580879 150 F150WW 0.622886 150 F150M M 0.625886 150 F150E E 0.727755 150 F150C C0.778063 150 F150I I 0.78431 150 F150K K 0.848249 153 I153N N 0.890296154 F154T T <0.01 154 F154D D <0.01 154 F154E E <0.01 154 F154G G <0.01154 F154L L <0.01 154 F154P P <0.01 154 F154V V <0.01 154 F154S S0.287767 154 F154Q Q 0.973299 194 I194S S <0.01 194 I194A A <0.01 194I194C C <0.01 194 I194P P <0.01 194 I194F F <0.01 194 I194W W <0.01 194I194R R <0.01 194 I194Y Y <0.01 194 I194G G 0.044503 194 I194L L0.577811 194 I194V V 0.780569 196 F196H H <0.01 196 F196G G <0.01 196F196S S <0.01 196 F196Q Q <0.01 196 F196A A <0.01 196 F196K K <0.01 196F196N N <0.01 196 F196R R <0.01 196 F196W W 0.38122 196 F196P P 0.385754196 F196V V 0.675769 196 F196M M 0.709899 196 F196Y Y 0.970105

The following Table provides variants that are better than wild-type atdegrading peracids (i.e., the performance index for the variant isbetter than the wild-type).

TABLE 10-6 Variants with Peracid Degradation Greater Than Wild-Type Pos.WT/Pos./Var. PAD PI 1 M001I 1.19 1 M001L 2.11 2 A002D 1.05 2 A002R 1.172 A002W 1.17 2 A002P 1.17 2 A002Q 1.29 2 A002E 1.38 3 K003T 1.03 3 K003S1.17 3 K003Q 1.19 3 K003R 1.29 3 K003Y 1.39 3 K003M 1.44 3 K003P 1.45 3K003C 1.52 3 K003L 1.84 3 K003H 1.89 3 K003A 2.14 3 K003I 2.44 3 K003E3.51 3 K003G 3.74 4 R004D 1.18 4 R004C 1.34 4 R004P 1.44 4 R004A 1.64 5I005M 1.09 5 I005E 1.59 5 I005L 1.63 5 I005A 1.88 5 I005C 2.47 5 I005D3.11 6 L006C 1.22 6 L006M 1.44 6 L006A 1.99 7 C007A 1.03 7 C007H 1.37 7C007I 1.48 7 C007E 1.63 7 C007K 2.95 8 F008M 1.11 8 F008L 1.31 8 F008A1.33 8 F008C 4.01 10 D010L 2.04 13 T013I 1.05 13 T013E 1.09 13 T013L1.47 13 T013M 1.47 13 T013C 1.55 13 T013A 1.88 13 T013N 2.61 13 T013P2.73 16 W016K 1.03 16 W016I 1.06 16 W016Y 1.09 16 W016L 1.16 17 V017S1.04 18 P018N 1.42 18 P018Q 3.26 18 P018R 3.97 18 P018C 4.16 18 P018Y4.17 18 P018V 4.85 18 P018E 4.87 18 P018G 4.96 18 P018H 6.05 18 P018L7.40 20 E020D 1.14 20 E020S 1.18 20 E020H 1.20 20 E020T 1.25 20 E020V1.27 20 E020A 1.28 20 E020W 1.30 20 E020N 1.34 20 E020P 1.43 20 E020Q1.56 20 E020C 1.76 21 D021S 1.11 21 D021E 1.39 21 D021F 1.41 21 D021W1.44 21 D021L 1.57 21 D021A 1.75 21 D021G 1.76 21 D021K 1.80 21 D021Y2.01 22 G022I 1.03 22 G022T 1.16 22 G022E 1.19 22 G022L 1.35 22 G022P1.36 22 G022Q 1.44 22 G022A 1.66 23 A023H 1.04 23 A023L 1.30 24 P024C1.04 24 P024K 1.36 24 P024L 1.51 26 E026M 1.10 26 E026H 1.19 26 E026D1.39 26 E026A 1.45 26 E026K 1.47 26 E026L 1.71 27 R027I 1.41 27 R027K1.55 27 R027L 2.60 27 R027A 2.78 28 F028E 1.04 28 F028W 1.17 28 F028C1.21 28 F028Y 1.36 28 F028M 1.37 28 F028A 1.48 28 F028L 2.02 28 F028D2.07 29 A029C 1.15 30 P030H 1.08 30 P030G 1.09 30 P030R 1.14 30 P030L1.17 30 P030E 1.24 30 P030Y 1.31 30 P030I 1.38 30 P030K 1.39 30 P030S1.49 30 P030T 1.64 30 P030V 1.74 31 D031V 1.08 31 D031T 1.11 31 D031Q1.13 31 D031W 1.14 31 D031G 1.16 31 D031A 1.18 31 D031S 1.23 31 D031F1.39 31 D031R 1.49 31 D031N 1.55 31 D031L 1.61 32 V032S 1.09 32 V032N1.61 32 V032W 1.71 32 V032Q 1.74 32 V032G 2.65 32 V032M 3.41 32 V032I3.51 32 V032A 3.64 32 V032E 3.92 32 V032D 4.19 32 V032L 4.72 32 V032K4.73 33 R033S 1.01 33 R033N 1.30 33 R033A 1.32 33 R033C 1.73 33 R033G2.63 33 R033K 2.72 33 R033L 2.90 34 W034P 1.21 34 W034M 1.22 34 W034C1.49 34 W034A 2.29 35 T035M 2.72 35 T035A 3.85 35 T035C 4.72 35 T035I5.38 35 T035E 5.73 36 G036C 1.06 36 G036A 1.07 36 G036H 1.10 36 G036K1.71 36 G036I 1.81 36 G036L 2.49 36 G036D 2.50 37 V037I 1.04 37 V037L1.16 37 V037S 1.49 37 V037N 1.52 37 V037C 1.63 37 V037A 2.00 37 V037P2.10 38 L038V 1.12 39 A039W 1.02 39 A039Y 1.13 40 Q040N 1.00 40 Q040I1.10 40 Q040E 1.28 40 Q040R 1.48 40 Q040L 1.49 40 Q040D 1.59 40 Q040S1.65 40 Q040T 1.81 40 Q040Y 2.02 40 Q040G 2.17 40 Q040W 2.59 40 Q040K3.64 41 Q041G 1.09 41 Q041H 1.14 41 Q041R 1.27 41 Q041K 1.61 41 Q041L1.92 41 Q041A 2.58 42 L042F 1.02 42 L042P 1.34 42 L042K 1.41 42 L042C1.43 43 G043A 1.07 43 G043L 1.82 43 G043E 1.88 44 A044C 1.92 45 D045F1.04 46 F046C 1.16 46 F046A 1.25 46 F046E 1.31 46 F046D 1.39 46 F046M1.42 46 F046K 1.46 46 F046P 1.50 46 F046L 1.54 47 E047L 1.02 47 E047K1.06 47 E047G 1.10 47 E047I 1.15 48 V048Q 1.39 48 V048F 1.42 48 V048A1.63 48 V048M 1.79 48 V048C 2.25 48 V048L 2.29 48 V048P 3.08 49 I049Y1.02 49 I049M 1.02 49 I049L 1.03 49 I049G 1.12 49 I049K 1.26 49 I049A1.87 50 E050P 1.02 50 E050M 1.04 50 E050G 1.11 50 E050D 1.22 50 E050A1.23 51 E051T 1.17 51 E051M 1.20 51 E051D 1.28 51 E051G 1.34 51 E051K2.00 51 E051A 2.72 52 G052W 2.47 53 L053H 1.70 54 S054N 1.29 54 S054P1.30 54 S054A 1.41 55 A055N 1.05 55 A055K 1.08 55 A055C 1.26 57 T057S1.01 57 T057G 1.05 58 T058L 1.12 58 T058H 1.49 59 N059Q 1.86 59 N059T5.63 59 N059S 7.32 59 N059K 8.21 59 N059E 9.88 59 N059V 9.97 59 N059G10.00 59 N059F 10.23 59 N059A 10.44 59 N059Y 11.14 59 N059C 11.23 59N059D 11.72 59 N059W 12.80 59 N059L 14.74 60 I060G 1.04 60 I060V 1.06 60I060H 1.07 60 I060Y 1.19 61 D061P 1.13 61 D061Q 1.16 61 D061L 1.20 61D061G 1.25 61 D061S 1.35 61 D061R 1.59 61 D061I 1.66 61 D061H 1.67 61D061K 1.72 63 P063K 1.02 63 P063V 1.04 63 P063Q 1.05 63 P063W 1.11 63P063G 1.22 63 P063L 1.23 63 P063T 1.32 64 T064G 1.08 64 T064M 1.09 64T064A 1.20 64 T064L 1.22 66 P066S 1.02 66 P066T 1.10 69 N069D 1.11 69N069A 1.13 69 N069Q 1.14 69 N069C 1.20 69 N069L 1.20 69 N069S 1.42 69N069T 1.43 69 N069H 1.52 69 N069K 1.59 69 N069V 1.73 69 N069I 1.75 70G070L 1.01 70 G070A 1.41 70 G070H 1.90 71 A071K 1.01 71 A071M 1.11 72S072F 1.15 72 S072G 1.76 72 S072M 2.13 72 S072C 2.18 72 S072H 2.48 72S072N 2.85 72 S072A 3.52 73 Y073M 1.13 73 Y073C 1.20 73 Y073A 1.40 74L074F 1.13 74 L074M 1.21 74 L074A 2.90 75 P075E 1.19 75 P075L 1.19 75P075W 1.31 75 P075Y 1.32 75 P075V 1.39 75 P075C 1.42 75 P075D 2.09 76S076C 1.06 76 S076T 1.11 76 S076A 1.11 76 S076H 1.11 76 S076P 1.20 76S076V 1.35 76 S076K 1.53 76 S076M 1.61 76 S076D 1.94 76 S076E 2.09 76S076G 2.15 76 S076L 4.70 77 C077T 1.03 77 C077D 1.05 78 L078T 1.10 78L078I 1.11 78 L078G 1.38 78 L078H 1.57 80 T080V 1.01 80 T080Q 1.07 80T080A 1.11 80 T080C 1.15 80 T080S 1.40 80 T080G 1.50 81 H081N 1.00 81H081L 1.03 81 H081W 1.09 81 H081C 1.09 81 H081A 1.45 81 H081M 1.54 82L082M 1.06 83 P083C 1.01 83 P083R 1.09 83 P083N 1.10 83 P083K 1.16 83P083E 1.26 83 P083M 1.88 83 P083A 2.36 84 L084F 1.01 84 L084G 1.01 85D085R 1.03 85 D085A 1.09 85 D085H 1.24 85 D085E 1.25 85 D085C 1.50 85D085G 1.60 85 D085F 1.98 86 L086C 2.44 86 L086A 3.32 87 V087P 1.64 87V087C 2.22 87 V087L 4.30 88 I088M 1.09 88 I088P 3.51 89 I089L 1.22 89I089A 1.83 89 I089P 1.91 90 M090C 1.09 90 M090E 1.15 90 M090A 1.41 90M090D 2.88 91 L091I 1.05 91 L091C 1.27 91 L091A 1.45 91 L091D 1.47 92G092C 2.05 93 T093A 1.05 96 T096F 1.24 96 T096G 1.28 96 T096L 1.93 96T096M 2.53 96 T096C 3.76 96 T096A 4.20 98 A098Y 1.15 98 A098P 1.26 98A098N 1.40 98 A098C 1.42 98 A098L 1.47 98 A098D 2.19 100 F100C 1.28 100F100T 1.42 100 F100N 1.45 100 F100A 2.02 100 F100M 2.19 101 R101L 1.12102 R102Q 1.19 102 R102Y 1.29 102 R102L 1.64 102 R102A 1.79 104 P104V1.02 104 P104H 1.03 104 P104N 1.44 104 P104C 1.83 104 P104E 1.97 104P104I 2.05 104 P104M 2.24 105 L105Q 1.04 105 L105H 1.23 105 L105R 1.25105 L105G 1.40 105 L105W 1.71 105 L105F 1.73 105 L105C 1.92 106 D106S1.02 106 D106W 1.07 106 D106E 1.09 106 D106C 1.10 106 D106A 1.13 106D106H 1.18 106 D106K 1.24 106 D106T 1.38 106 D106F 1.45 106 D106G 1.45106 D106V 1.68 107 I107L 1.04 107 I107S 1.33 107 I107C 1.41 107 I107T1.53 108 A108S 1.00 108 A108G 1.13 108 A108L 2.56 108 A108K 2.97 110G110A 1.01 110 G110D 1.40 110 G110C 1.43 110 G110E 1.76 110 G110F 2.29111 M111C 1.01 111 M111A 1.02 111 M111I 1.03 111 M111Y 1.06 111 M111W1.23 111 M111N 1.31 112 S112L 1.00 112 S112E 1.16 113 V113M 1.06 113V113Q 1.11 113 V113R 1.11 113 V113P 1.14 113 V113N 1.22 113 V113A 1.31114 L114T 1.05 114 L114A 1.07 114 L114G 1.14 114 L114C 1.14 114 L114I1.17 114 L114M 1.28 115 V115C 1.08 115 V115S 1.14 115 V115Q 1.15 115V115A 1.19 115 V115T 1.28 115 V115L 1.30 115 V115M 1.32 115 V115R 1.63115 V115F 1.69 115 V115G 1.76 115 V115Y 2.07 115 V115D 2.21 115 V115P2.21 115 V115W 2.48 116 T116N 1.05 116 T116C 1.05 116 T116H 1.08 116T116M 1.39 117 Q117F 1.02 117 Q117R 1.05 117 Q117T 1.10 117 Q117H 1.12117 Q117Y 1.13 117 Q117P 1.13 117 Q117E 1.21 117 Q117A 1.73 117 Q117M1.89 118 V118L 1.05 118 V118C 1.14 118 V118Y 1.34 118 V118Q 1.50 119L119A 1.02 120 T120V 1.07 120 T120S 1.07 120 T120K 1.09 120 T120M 1.22120 T120L 1.26 120 T120N 1.42 120 T120E 1.53 120 T120I 1.56 120 T120Y1.61 121 S121E 1.04 121 S121N 1.06 121 S121Q 1.09 121 S121T 1.26 121S121L 1.49 121 S121A 1.55 121 S121V 1.59 121 S121C 1.64 122 A122L 1.02123 G123K 1.12 123 G123A 1.19 123 G123Y 1.24 123 G123M 1.38 123 G123L1.38 123 G123W 1.39 125 V125G 1.09 126 G126M 1.17 126 G126D 1.22 127T127A 1.10 128 T128M 1.06 128 T128H 1.08 128 T128V 1.15 128 T128P 1.16128 T128W 1.23 128 T128S 1.27 128 T128A 1.31 128 T128Q 1.34 128 T128N1.36 128 T128K 1.57 128 T128R 1.70 128 T128F 1.71 128 T128L 1.72 128T128Y 1.81 131 A131R 1.04 132 P132N 1.05 132 P132L 2.24 132 P132E 3.02132 P132Y 4.78 132 P132G 4.98 132 P132S 5.05 132 P132C 5.68 132 P132A6.08 132 P132Q 6.15 133 K133Y 1.44 133 K133L 1.92 134 V134C 1.37 134V134G 1.42 134 V134S 1.44 134 V134L 1.45 134 V134A 1.64 134 V134P 1.71134 V134M 1.89 134 V134N 2.80 135 L135D 2.90 136 V136T 1.13 136 V136L1.13 136 V136C 1.23 136 V136A 1.60 137 V137M 1.13 137 V137L 1.27 137V137C 1.42 137 V137A 1.46 138 S138G 1.11 138 S138C 1.18 138 S138A 1.28138 S138N 1.31 138 S138P 1.39 140 P140C 1.07 140 P140A 1.83 140 P140H2.25 140 P140F 2.89 140 P140G 3.11 141 P141A 1.08 143 A143C 1.07 143A143E 1.13 143 A143D 1.22 143 A143L 1.28 143 A143H 1.36 143 A143K 1.37144 P144M 1.01 144 P144F 1.08 144 P144Q 1.08 144 P144K 1.09 144 P144R1.14 144 P144L 1.15 144 P144D 1.38 144 P144N 1.49 144 P144H 1.60 144P144Y 1.65 146 P146N 1.00 146 P146G 1.04 146 P146R 1.06 146 P146M 1.23146 P146A 1.36 146 P146Y 1.44 146 P146F 1.53 146 P146H 1.57 146 P146C1.69 146 P146L 2.00 147 H147Q 1.03 147 H147W 1.05 147 H147K 1.06 147H147E 1.10 147 H147Y 1.12 147 H147C 1.17 147 H147D 1.18 147 H147P 1.21147 H147N 1.25 147 H147L 1.29 147 H147M 1.44 148 P148V 1.04 148 P148A1.06 148 P148T 1.09 148 P148E 1.19 148 P148G 1.20 148 P148S 1.21 148P148R 1.25 148 P148K 1.30 148 P148D 1.34 148 P148Y 1.37 148 P148L 1.39148 P148F 1.50 149 W149H 1.01 150 F150Y 1.07 150 F150H 1.18 150 F150L1.30 151 Q151P 1.91 151 Q151E 2.07 151 Q151K 2.19 151 Q151H 2.19 151Q151S 2.25 151 Q151R 2.32 151 Q151T 2.37 151 Q151C 2.55 151 Q151Y 2.75151 Q151D 2.81 151 Q151A 2.93 151 Q151M 6.36 152 L152M 1.10 152 L152C1.14 152 L152E 1.23 152 L152A 1.29 152 L152Y 1.37 152 L152W 1.55 153I153V 1.15 153 I153A 1.49 153 I153L 1.50 153 I153T 1.62 153 I153S 1.66153 I153F 1.75 153 I153P 1.87 153 I153H 2.00 153 I153K 2.44 154 F154Y4.96 155 E155S 1.12 155 E155G 1.12 155 E155T 1.19 155 E155D 1.24 155E155K 1.33 155 E155N 1.79 155 E155L 2.07 155 E155A 2.59 155 E155P 2.60155 E155Y 2.65 155 E155M 2.91 156 G156S 1.04 156 G156K 1.11 156 G156E1.14 156 G156R 1.21 156 G156A 1.21 156 G156P 1.29 156 G156C 1.37 156G156N 1.38 156 G156H 1.40 156 G156Y 1.40 156 G156T 1.53 156 G156M 1.62156 G156D 1.62 157 G157I 1.33 157 G157F 1.42 157 G157K 1.47 157 G157H1.57 158 E158H 1.01 158 E158P 1.19 158 E158Q 1.24 158 E158S 1.27 158E158A 1.28 158 E158R 1.29 158 E158W 1.31 158 E158C 1.37 158 E158N 1.58158 E158M 1.73 158 E158F 1.77 158 E158K 1.88 158 E158L 1.96 158 E158Y2.48 159 Q159H 1.48 160 K160N 1.12 160 K160A 1.14 160 K160R 1.15 160K160D 1.19 160 K160C 1.29 160 K160Q 1.41 160 K160M 1.47 160 K160P 1.66161 T161L 1.16 161 T161V 1.24 161 T161Q 1.50 161 T161M 1.72 161 T161Y2.62 162 T162R 1.23 162 T162G 1.82 162 T162S 2.01 162 T162W 2.04 162T162I 2.21 162 T162Q 2.45 162 T162Y 2.89 162 T162K 3.13 162 T162F 3.23162 T162M 3.49 162 T162C 3.57 162 T162L 3.59 162 T162N 3.84 162 T162H3.91 162 T162P 4.37 163 E163N 1.00 163 E163C 1.08 163 E163D 1.08 163E163A 1.79 163 E163Y 1.89 163 E163L 1.94 164 L164Q 1.01 164 L164V 1.02164 L164S 1.11 164 L164M 1.26 164 L164N 1.31 164 L164R 1.61 164 L164P2.41 165 A165G 1.07 165 A165V 1.13 165 A165N 1.20 165 A165R 1.29 165A165Q 1.32 165 A165T 1.32 165 A165P 1.34 165 A165C 1.42 165 A165L 1.55165 A165M 1.56 165 A165D 1.69 166 R166W 1.08 166 R166F 1.10 166 R166K1.20 166 R166N 1.21 166 R166Y 1.22 166 R166M 1.29 166 R166I 1.39 166R166P 1.50 166 R166L 1.50 166 R166A 1.51 166 R166D 1.55 166 R166H 1.56167 V167I 1.00 167 V167S 1.86 167 V167H 2.11 167 V167Y 2.15 167 V167R2.25 167 V167Q 2.41 167 V167T 2.47 167 V167L 2.56 167 V167G 2.83 167V167M 3.84 167 V167A 4.99 167 V167C 5.37 167 V167D 5.54 167 V167P 6.08168 Y168F 5.17 168 Y168L 5.39 169 S169Y 1.10 169 S169A 1.13 169 S169R1.19 169 S169K 1.27 169 S169Q 1.37 169 S169C 1.38 169 S169M 1.40 169S169L 1.47 169 S169I 1.53 170 A170C 1.06 170 A170E 1.17 170 A170F 1.17170 A170N 1.17 170 A170M 1.28 170 A170D 1.32 170 A170P 1.33 171 L171H1.07 171 L171G 1.33 171 L171Y 1.35 171 L171T 1.36 171 L171V 1.39 171L171I 1.42 171 L171K 1.53 171 L171A 1.66 171 L171C 1.73 171 L171S 1.76171 L171Q 1.93 171 L171F 1.97 171 L171M 2.22 171 L171N 2.79 172 A172M1.06 172 A172L 1.22 172 A172D 1.42 172 A172Y 1.76 173 S173T 1.29 173S173H 1.49 173 S173I 2.22 173 S173F 2.30 173 S173R 2.47 173 S173V 2.54173 S173E 2.65 173 S173P 2.66 173 S173A 2.72 173 S173M 3.01 173 S173K3.01 173 S173C 3.07 173 S173Y 3.54 173 S173W 3.67 173 S173L 3.86 174F174H 1.05 174 F174K 1.17 174 F174P 1.46 174 F174Y 1.66 174 F174L 1.83174 F174A 2.09 174 F174M 2.20 175 M175N 1.02 175 M175E 1.43 176 K176C1.01 176 K176R 1.03 176 K176E 1.08 176 K176W 1.16 176 K176D 1.18 176K176A 1.19 176 K176F 1.28 176 K176V 1.33 176 K176M 1.33 178 P178K 1.70178 P178T 2.28 178 P178V 2.70 178 P178G 2.95 178 P178S 3.06 178 P178Q3.64 178 P178M 3.87 178 P178E 4.15 178 P178A 4.39 178 P178D 6.44 178P178Y 6.91 178 P178L 7.15 179 F179G 1.16 179 F179V 1.17 179 F179Y 1.47179 F179E 1.80 179 F179L 1.89 180 F180W 1.81 180 F180C 1.94 180 F180I2.11 180 F180L 2.13 180 F180A 2.70 180 F180Y 2.99 180 F180N 3.05 180F180V 3.24 180 F180M 4.36 181 D181A 1.23 183 G183P 1.02 183 G183R 1.09183 G183Y 1.45 183 G183L 1.50 183 G183C 1.99 184 S184Y 1.09 184 S184Q1.16 184 S184I 1.21 184 S184V 1.25 184 S184F 1.27 184 S184K 1.61 184S184A 1.69 184 S184M 1.77 184 S184E 1.86 184 S184N 1.93 184 S184L 2.00184 S184D 2.24 184 S184C 2.39 185 V185F 1.20 185 V185Q 1.41 185 V185M1.46 186 I186L 1.14 186 I186M 1.38 186 I186A 1.79 186 I186D 4.29 187S187K 1.16 187 S187D 1.40 187 S187G 1.46 187 S187L 1.46 187 S187H 1.51187 S187I 1.58 187 S187N 1.59 187 S187C 1.67 187 S187A 1.72 187 S187M1.87 188 T188N 1.69 188 T188E 1.97 189 D189A 1.18 189 D189T 1.21 189D189I 1.27 189 D189L 1.30 190 G190C 1.17 190 G190Y 1.39 190 G190P 1.86190 G190D 2.02 190 G190H 2.92 190 G190A 3.42 190 G190M 5.54 191 V191T1.03 191 V191R 1.91 191 V191K 2.17 191 V191F 2.75 191 V191C 2.81 191V191Y 4.34 191 V191L 4.69 191 V191A 5.06 191 V191E 5.46 191 V191Q 5.83191 V191D 6.03 191 V191M 7.34 193 G193S 1.60 193 G193E 3.15 193 G193Q4.29 193 G193V 5.21 195 H195P 1.16 195 H195M 1.28 195 H195K 1.33 195H195Y 1.49 195 H195E 1.70 195 H195D 1.93 196 F196I 1.12 196 F196L 1.17196 F196C 1.18 197 T197H 1.24 197 T197A 1.42 197 T197M 2.38 198 E198T1.16 198 E198S 1.18 198 E198F 1.21 198 E198V 1.44 198 E198Q 1.46 198E198A 1.46 198 E198I 1.48 198 E198L 1.54 198 E198N 1.67 198 E198P 1.72198 E198Y 1.77 198 E198W 1.78 198 E198C 1.83 198 E198M 1.86 198 E198R1.88 199 A199F 1.15 199 A199H 1.15 199 A199R 1.17 199 A199T 1.22 199A199E 1.31 199 A199D 1.33 199 A199V 1.45 199 A199K 1.53 199 A199Y 1.59199 A199L 1.65 199 A199C 2.45 201 N201D 1.64 202 R202M 1.76 202 R202G1.82 202 R202S 1.84 202 R202C 1.93 202 R202A 1.97 202 R202I 1.99 202R202E 2.05 202 R202L 2.05 202 R202T 2.06 202 R202H 2.09 202 R202F 2.16202 R202W 2.52 203 D203Q 1.03 203 D203S 1.13 203 D203I 1.19 203 D203N1.28 203 D203G 1.33 203 D203F 1.34 203 D203H 1.54 203 D203P 1.71 203D203R 1.77 203 D203A 1.96 203 D203L 2.08 203 D203C 2.09

The following Table provides variants that exhibited peracid degradationthat was less than wild-type.

TABLE 10-7 Variants with Peracid Degradation Results Less than Wild-TypePos WT/Pos./Var. PAD PI 1 M001V 0.94 2 A002Y 0.46 2 A002N 0.59 2 A002V0.60 2 A002I 0.61 2 A002T 0.61 2 A002S 0.66 2 A002G 0.84 2 A002F 0.93 3K003V 0.84 4 R004L 0.01 4 R004V 0.08 4 R004I 0.15 4 R004W 0.48 4 R004G0.79 4 R004S 0.91 4 R004E 0.97 4 R004Y 0.98 4 R004H 0.99 4 R004Q 0.99 4R004T 1.00 5 I005G 0.01 5 I005N 0.01 5 I005P 0.01 5 I005R 0.01 5 I005F0.15 5 I005S 0.37 5 I005H 0.63 5 I005T 0.72 5 I005V 0.92 6 L006S 0.01 6L006K 0.01 6 L006G 0.01 6 L006H 0.01 6 L006R 0.01 6 L006W 0.01 6 L006E0.01 6 L006Q 0.01 6 L006V 0.35 6 L006T 0.35 6 L006I 0.82 7 C007S 0.01 7C007R 0.01 7 C007Y 0.54 7 C007M 0.68 7 C007G 0.69 8 F008S 0.01 8 F008R0.46 8 F008H 0.64 8 F008G 0.65 8 F008T 0.77 8 F008K 0.83 8 F008P 0.83 8F008V 0.85 8 F008Y 0.90 8 F008N 0.96 9 G009H 0.01 9 G009T 0.01 10 D010W0.01 10 D010K 0.01 10 D010Y 0.01 10 D010T 0.01 10 D010I 0.01 10 D010V0.01 10 D010S 0.01 10 D010G 0.01 10 D010R 0.01 10 D010A 0.01 10 D010M0.01 10 D010N 0.01 10 D010P 0.01 10 D010E 0.15 11 S011T 0.01 11 S011V0.01 11 S011D 0.01 11 S011E 0.01 11 S011F 0.01 11 S011G 0.01 11 S011L0.01 11 S011Q 0.01 11 S011R 0.01 11 S011H 0.33 11 S011K 0.40 11 S011A0.53 11 S011I 0.56 12 L012V 0.01 12 L012S 0.01 12 L012G 0.01 12 L012R0.01 12 L012D 0.01 12 L012P 0.01 12 L012W 0.02 12 L012T 0.06 12 L012A0.07 12 L012K 0.13 12 L012H 0.16 12 L012F 0.17 12 L012Q 0.22 12 L012C0.22 12 L012N 0.66 13 T013Q 0.51 13 T013V 0.63 13 T013S 0.68 13 T013G0.77 14 W014I 0.01 14 W014S 0.01 14 W014G 0.01 14 W014K 0.01 14 W014V0.01 14 W014L 0.01 14 W014T 0.01 14 W014R 0.01 14 W014N 0.01 14 W014P0.01 14 W014E 0.15 14 W014F 0.22 14 W014A 0.27 14 W014Y 0.66 15 G015C0.01 15 G015N 0.01 15 G015D 0.01 15 G015E 0.01 15 G015P 0.01 15 G015A0.61 15 G015S 0.63 16 W016S 0.01 16 W016G 0.01 16 W016H 0.01 16 W016T0.01 16 W016R 0.01 16 W016N 0.01 16 W016P 0.15 16 W016Q 0.31 16 W016M0.37 16 W016A 0.55 16 W016D 0.57 16 W016E 0.65 16 W016V 0.88 17 V017A0.68 17 V017E 0.75 17 V017G 0.84 17 V017K 0.84 17 V017F 0.85 17 V017T0.86 17 V017Y 0.88 17 V017R 0.94 17 V017P 0.96 17 V017I 0.99 17 V017L1.00 18 P018S 0.07 19 V019P 0.01 19 V019M 0.12 19 V019R 0.34 19 V019Q0.40 19 V019A 0.55 19 V019G 0.56 19 V019S 0.57 19 V019E 0.62 19 V019Y0.70 19 V019D 0.79 19 V019L 0.91 19 V019K 0.97 20 E020L 0.73 20 E020G0.78 21 D021P 0.86 22 G022K 0.01 22 G022W 0.23 22 G022R 0.56 22 G022V0.85 22 G022S 0.98 23 A023R 0.28 23 A023S 0.34 23 A023G 0.35 23 A023F0.44 23 A023V 0.60 23 A023Q 0.73 23 A023P 0.73 23 A023W 0.80 23 A023M0.95 23 A023Y 0.96 24 P024S 0.61 24 P024Q 0.65 24 P024T 0.66 24 P024A0.68 24 P024G 0.76 24 P024I 0.85 24 P024R 0.91 24 P024H 0.97 25 T025P0.01 25 T025H 0.01 25 T025L 0.01 25 T025R 0.01 25 T025M 0.01 25 T025E0.01 25 T025D 0.01 25 T025K 0.13 25 T025W 0.14 25 T025I 0.35 25 T025G0.43 25 T025C 0.51 25 T025V 0.51 25 T025S 0.58 25 T025A 0.86 26 E026S0.28 26 E026T 0.40 26 E026W 0.47 26 E026N 0.48 26 E026R 0.81 26 E026G0.87 26 E026C 0.94 26 E026V 0.97 26 E026P 0.99 27 R027W 0.01 27 R027T0.01 27 R027P 0.48 27 R027C 0.58 27 R027S 0.69 27 R027G 0.84 27 R027E0.93 27 R027V 0.94 28 F028G 0.01 28 F028P 0.39 28 F028V 0.53 28 F028S0.70 29 A029V 0.44 29 A029T 0.47 29 A029S 0.55 29 A029Y 0.59 29 A029P0.62 29 A029R 0.73 29 A029W 0.74 29 A029M 0.77 29 A029G 0.80 29 A029E0.84 29 A029D 1.00 30 P030M 0.79 30 P030Q 0.91 30 P030A 0.92 31 D031E0.88 32 V032P 0.01 32 V032R 0.72 33 R033V 0.94 34 W034R 0.01 34 W034E0.01 34 W034Q 0.04 34 W034S 0.08 34 W034T 0.15 34 W034V 0.73 34 W034G0.88 34 W034I 0.94 35 T035Q 0.01 35 T035N 0.01 35 T035R 0.01 35 T035V0.34 36 G036S 0.26 36 G036T 0.33 36 G036V 0.38 36 G036M 0.54 36 G036N0.56 36 G036W 0.68 36 G036Q 0.71 36 G036R 0.90 37 V037T 0.81 37 V037H0.96 37 V037W 0.98 38 L038K 0.01 38 L038G 0.01 38 L038E 0.01 38 L038P0.01 38 L038Q 0.01 38 L038R 0.01 38 L038D 0.12 38 L038S 0.29 38 L038A0.63 38 L038C 0.72 39 A039S 0.01 39 A039G 0.30 39 A039N 0.43 39 A039R0.64 39 A039I 0.71 39 A039P 0.74 39 A039T 0.79 39 A039M 0.81 39 A039E0.83 39 A039C 0.92 39 A039K 0.96 39 A039L 0.97 39 A039V 0.98 40 Q040P0.01 41 Q041V 0.01 41 Q041S 0.22 41 Q041P 0.66 41 Q041Y 0.70 41 Q041W0.88 42 L042W 0.01 42 L042H 0.01 42 L042T 0.01 42 L042Q 0.28 42 L042S0.45 42 L042R 0.64 42 L042I 0.66 42 L042V 0.73 42 L042M 0.74 42 L042G0.76 43 G043S 0.23 43 G043P 0.31 43 G043V 0.33 43 G043Q 0.48 43 G043R0.59 43 G043C 0.73 43 G043I 0.77 43 G043K 0.86 43 G043M 0.88 43 G043Y0.94 43 G043H 0.96 44 A044S 0.01 44 A044Y 0.01 44 A044T 0.01 44 A044R0.01 44 A044E 0.03 44 A044V 0.50 44 A044F 0.80 44 A044W 0.85 44 A044M0.98 44 A044L 0.99 45 D045S 0.38 45 D045T 0.44 45 D045R 0.49 45 D045V0.50 45 D045P 0.53 45 D045Q 0.57 45 D045W 0.58 45 D045H 0.78 45 D045L0.78 45 D045M 0.78 45 D045G 0.84 45 D045A 0.84 45 D045C 0.84 45 D045K0.87 46 F046T 0.43 46 F046W 0.63 46 F046S 0.66 46 F046V 0.79 46 F046I0.88 46 F046G 0.94 47 E047P 0.36 47 E047R 0.62 47 E047N 0.63 47 E047S0.63 47 E047M 0.70 47 E047A 0.76 47 E047F 0.76 47 E047C 0.77 47 E047T0.84 47 E047D 0.98 47 E047H 0.99 48 V048R 0.01 48 V048S 0.42 48 V048G0.87 48 V048N 0.98 48 V048E 0.99 49 I049P 0.16 49 I049R 0.29 49 I049W0.68 49 I049H 0.74 49 I049S 0.79 49 I049E 0.88 49 I049V 0.97 50 E050R0.01 50 E050W 0.14 50 E050V 0.43 50 E050I 0.58 50 E050S 0.65 50 E050Q0.91 50 E050L 0.97 51 E051R 0.01 51 E051I 0.04 51 E051W 0.17 51 E051V0.37 51 E051Q 0.76 51 E051L 0.93 52 G052H 0.01 52 G052S 0.01 52 G052V0.01 52 G052T 0.01 52 G052M 0.01 52 G052F 0.01 52 G052I 0.07 52 G052P0.24 52 G052L 0.24 52 G052Q 0.28 52 G052R 0.35 52 G052E 0.55 52 G052A0.79 53 L053R 0.01 53 L053W 0.01 53 L053P 0.01 53 L053D 0.01 53 L053E0.19 53 L053K 0.24 53 L053S 0.26 53 L053G 0.33 53 L053V 0.65 53 L053I0.66 53 L053Q 0.72 53 L053T 0.84 54 S054F 0.01 54 S054W 0.01 54 S054H0.01 54 S054K 0.08 54 S054I 0.12 54 S054Y 0.12 54 S054G 0.17 54 S054L0.26 54 S054V 0.29 54 S054E 0.30 54 S054T 0.33 54 S054R 0.35 54 S054M0.48 54 S054Q 0.53 54 S054D 0.65 54 S054C 0.88 55 A055V 0.01 55 A055I0.01 55 A055P 0.01 55 A055W 0.01 55 A055Y 0.18 55 A055R 0.25 55 A055T0.42 55 A055G 0.73 55 A055L 0.87 55 A055S 0.87 55 A055H 0.92 56 R056C0.01 56 R056G 0.01 56 R056T 0.01 56 R056E 0.01 56 R056Q 0.01 56 R056S0.12 56 R056L 0.24 56 R056N 0.27 56 R056A 0.69 57 T057R 0.01 57 T057P0.01 57 T057N 0.25 57 T057C 0.40 57 T057Y 0.55 57 T057H 0.61 57 T057A0.65 57 T057L 0.76 57 T057V 0.87 57 T057I 0.87 58 T058M 0.03 58 T058A0.36 58 T058V 0.96 58 T058S 0.96 59 N059R 0.01 59 N059M 0.01 59 N059P0.01 60 I060P 0.32 60 I060D 0.66 60 I060C 0.67 60 I060M 0.68 60 I060A0.79 60 I060R 0.81 60 I060L 0.91 60 I060E 0.92 60 I060K 0.96 60 I060S1.00 61 D061F 0.70 61 D061A 0.71 61 D061C 0.85 61 D061Y 0.95 61 D061V0.97 61 D061N 1.00 62 D062T 0.01 62 D062I 0.01 62 D062V 0.01 62 D062H0.01 62 D062W 0.01 62 D062S 0.01 62 D062L 0.01 62 D062G 0.01 62 D062R0.01 62 D062M 0.01 62 D062P 0.01 62 D062Q 0.01 62 D062A 0.11 62 D062C0.49 62 D062E 0.60 63 P063A 0.60 63 P063R 0.80 63 P063S 0.90 63 P063M0.91 63 P063F 0.93 63 P063Y 0.95 64 T064R 0.11 64 T064D 0.64 64 T064W0.69 64 T064Q 0.87 64 T064C 0.88 64 T064P 0.94 64 T064H 0.96 64 T064N0.98 64 T064S 0.99 65 D065V 0.20 65 D065R 0.22 65 D065H 0.40 65 D065Y0.42 65 D065P 0.42 65 D065S 0.47 65 D065W 0.50 65 D065T 0.50 65 D065G0.52 65 D065I 0.62 65 D065A 0.72 66 P066N 0.38 66 P066Q 0.42 66 P066G0.44 66 P066R 0.51 66 P066C 0.52 66 P066A 0.56 66 P066F 0.67 66 P066Y0.70 66 P066D 0.72 66 P066I 0.84 66 P066V 0.89 66 P066H 0.95 66 P066L0.99 67 R067F 0.01 67 R067W 0.02 67 R067P 0.04 67 R067E 0.11 67 R067V0.12 67 R067Q 0.13 67 R067L 0.16 67 R067A 0.22 67 R067T 0.32 67 R067N0.33 67 R067G 0.41 67 R067K 0.99 68 L068G 0.01 68 L068A 0.01 68 L068M0.03 68 L068C 0.06 68 L068S 0.07 68 L068N 0.10 68 L068E 0.13 68 L068H0.22 68 L068Q 0.25 68 L068F 0.25 68 L068T 0.32 68 L068P 0.35 68 L068D0.44 68 L068Y 0.45 68 L068R 0.47 68 L068V 0.51 68 L068W 0.56 68 L068I0.73 69 N069Y 0.17 69 N069W 0.55 69 N069P 0.59 69 N069R 0.83 69 N069G0.98 70 G070M 0.01 70 G070T 0.01 70 G070P 0.01 70 G070V 0.01 70 G070C0.01 70 G070R 0.01 70 G070Y 0.01 70 G070K 0.01 70 G070N 0.01 70 G070Q0.01 70 G070F 0.01 70 G070I 0.27 70 G070E 0.33 70 G070S 0.64 71 A071P0.01 71 A071N 0.61 71 A071D 0.65 71 A071G 0.68 71 A071S 0.69 71 A071R0.77 71 A071H 0.78 71 A071I 0.79 71 A071T 0.79 71 A071E 0.81 71 A071L0.84 71 A071F 0.99 71 A071C 0.99 72 S072Y 0.07 72 S072W 0.34 72 S072P0.56 72 S072Q 0.66 72 S072L 0.70 72 S072R 0.74 72 S072D 0.80 72 S072V0.83 72 S072E 0.93 72 S072T 0.97 73 Y073P 0.01 73 Y073R 0.26 73 Y073L0.50 73 Y073G 0.51 73 Y073H 0.52 73 Y073I 0.64 73 Y073S 0.68 73 Y073V0.74 73 Y073N 0.76 73 Y073D 0.80 73 Y073Q 0.87 73 Y073K 0.94 74 L074S0.01 74 L074G 0.57 74 L074V 0.61 74 L074I 0.64 74 L074W 0.67 74 L074Y0.86 75 P075M 0.30 75 P075R 0.46 75 P075Q 0.61 75 P075S 0.63 75 P075T0.69 75 P075I 0.74 75 P075H 0.86 75 P075K 0.88 75 P075G 0.93 76 S076W0.01 76 S076Y 0.18 76 S076F 0.46 76 S076Q 0.90 77 C077Y 0.01 77 C077R0.01 77 C077W 0.01 77 C077F 0.01 77 C077G 0.18 77 C077L 0.73 77 C077S0.76 77 C077V 0.80 77 C077A 0.91 78 L078E 0.01 78 L078N 0.01 78 L078M0.48 78 L078Q 0.52 78 L078C 0.78 78 L078Y 0.81 78 L078V 0.83 79 A079H0.01 79 A079F 0.01 79 A079C 0.03 79 A079Q 0.27 79 A079E 0.27 79 A079N0.28 79 A079M 0.28 79 A079R 0.32 79 A079W 0.53 79 A079T 0.60 79 A079I0.67 79 A079S 0.78 79 A079G 0.92 79 A079P 0.94 79 A079L 0.96 80 T080W0.01 80 T080L 0.01 80 T080K 0.01 80 T080R 0.01 80 T080E 0.01 80 T080P0.01 80 T080H 0.05 80 T080Y 0.11 80 T080I 0.15 80 T080N 0.53 81 H081R0.01 81 H081Y 0.14 81 H081K 0.56 81 H081S 0.69 81 H081V 0.71 81 H081P0.72 81 H081Q 0.75 81 H081G 0.80 81 H081F 0.90 82 L082R 0.01 82 L082S0.01 82 L082W 0.01 82 L082V 0.19 82 L082G 0.31 82 L082T 0.38 82 L082H0.47 82 L082I 0.51 82 L082K 0.51 82 L082P 0.52 82 L082A 0.98 83 P083T0.01 83 P083V 0.19 83 P083L 0.21 83 P083H 0.61 83 P083W 0.62 83 P083G0.68 83 P083S 0.79 83 P083Q 0.82 83 P083D 0.83 83 P083F 0.99 84 L084W0.01 84 L084V 0.42 84 L084P 0.43 84 L084T 0.44 84 L084A 0.45 84 L084Q0.52 84 L084S 0.55 84 L084R 0.57 84 L084N 0.67 84 L084K 0.79 84 L084D0.85 84 L084I 0.87 84 L084H 0.99 85 D085I 0.10 85 D085L 0.24 85 D085V0.25 85 D085W 0.34 85 D085P 0.54 85 D085Y 0.55 85 D085S 0.68 85 D085T0.71 85 D085N 0.78 85 D085Q 0.99 86 L086H 0.01 86 L086S 0.01 86 L086R0.01 86 L086E 0.01 86 L086Q 0.01 86 L086W 0.08 86 L086V 0.12 86 L086T0.28 86 L086G 0.70 86 L086Y 0.82 86 L086P 0.99 87 V087S 0.01 87 V087G0.01 87 V087Y 0.01 87 V087R 0.01 87 V087K 0.01 87 V087D 0.01 87 V087F0.10 87 V087T 0.15 87 V087A 0.17 87 V087M 0.75 88 I088H 0.01 88 I088T0.01 88 I088G 0.01 88 I088N 0.01 88 I088Q 0.01 89 I089H 0.01 89 I089S0.01 89 I089G 0.01 89 I089W 0.01 89 I089Q 0.01 89 I089E 0.01 89 I089F0.75 89 I089V 0.82 89 I089T 0.90 90 M090S 0.01 90 M090W 0.01 90 M090G0.01 90 M090P 0.01 90 M090V 0.08 90 M090T 0.15 90 M090R 0.36 90 M090I0.66 90 M090Q 0.77 90 M090L 0.98 91 L091G 0.01 91 L091T 0.01 91 L091Q0.01 91 L091E 0.01 91 L091S 0.43 91 L091V 0.79 91 L091M 0.88 92 G092V0.01 92 G092S 0.01 92 G092E 0.01 92 G092F 0.01 93 T093Q 0.01 93 T093Y0.03 93 T093D 0.23 93 T093S 0.49 93 T093F 0.54 93 T093C 0.95 94 N094L0.01 94 N094T 0.01 94 N094V 0.01 94 N094H 0.01 94 N094R 0.01 94 N094W0.01 94 N094M 0.03 94 N094C 0.07 94 N094Y 0.12 94 N094G 0.53 94 N094A0.74 94 N094P 0.79 94 N094S 0.88 95 D095E 0.75 96 T096I 0.01 96 T096W0.01 96 T096Y 0.01 96 T096R 0.14 96 T096V 0.59 96 T096S 0.79 96 T096P0.89 97 K097Q 0.01 97 K097G 0.01 97 K097I 0.01 97 K097W 0.01 97 K097L0.01 97 K097V 0.01 97 K097Y 0.01 97 K097S 0.01 97 K097T 0.01 97 K097M0.22 97 K097A 0.23 97 K097P 0.27 97 K097R 0.59 98 A098T 0.27 98 A098G0.56 98 A098S 0.65 98 A098I 0.65 98 A098H 0.92 99 Y099R 0.29 99 Y099V0.31 99 Y099S 0.37 99 Y099W 0.57 99 Y099H 0.59 99 Y099I 0.61 99 Y099G0.70 99 Y099P 0.81 99 Y099A 0.82 99 Y099L 0.86 100 F100W 0.01 100 F100K0.01 100 F100D 0.01 100 F100E 0.15 100 F100S 0.85 101 R101W 0.01 101R101K 0.07 101 R101Q 0.11 101 R101V 0.44 101 R101D 0.80 101 R101Y 0.80101 R101P 0.86 101 R101N 0.92 101 R101C 0.95 101 R101I 0.96 101 R101F0.97 102 R102W 0.01 102 R102F 0.23 102 R102G 0.27 102 R102C 0.36 102R102V 0.61 102 R102D 0.68 102 R102P 0.89 102 R102S 0.96 103 T103W 0.01103 T103Y 0.01 103 T103G 0.01 103 T103K 0.01 103 T103I 0.01 103 T103L0.01 103 T103H 0.01 103 T103A 0.01 103 T103V 0.01 103 T103S 0.01 103T103C 0.01 103 T103R 0.01 103 T103N 0.01 103 T103F 0.01 103 T103P 0.01104 P104R 0.01 104 P104W 0.23 104 P104T 0.33 104 P104S 0.53 104 P104Q0.85 104 P104F 0.86 104 P104G 0.98 105 L105V 0.01 105 L105E 0.53 105L105S 0.61 105 L105Y 0.62 105 L105T 0.64 105 L105P 0.90 106 D106R 0.56106 D106Q 0.62 106 D106P 0.63 106 D106N 0.64 106 D106M 0.86 106 D106I0.92 106 D106L 1.00 107 I107E 0.01 107 I107G 0.01 107 I107F 0.01 107I107Q 0.01 107 I107R 0.01 107 I107P 0.32 107 I107Y 0.52 107 I107A 0.80107 I107N 0.93 107 I107V 0.97 108 A108E 0.61 108 A108Q 0.73 108 A108T0.87 108 A108V 0.95 109 L109W 0.01 109 L109D 0.11 109 L109I 0.14 109L109E 0.19 109 L109R 0.21 109 L109H 0.22 109 L109Q 0.22 109 L109F 0.32109 L109A 0.32 109 L109S 0.38 109 L109P 0.43 109 L109G 0.51 109 L109V0.54 109 L109M 0.63 109 L109N 0.66 109 L109T 0.79 109 L109Y 0.83 110G110T 0.01 110 G110W 0.01 110 G110Y 0.01 110 G110P 0.22 110 G110I 0.23110 G110S 0.30 110 G110Q 0.34 110 G110R 0.48 110 G110H 0.73 110 G110N0.77 110 G110M 0.82 111 M111R 0.01 111 M111S 0.14 111 M111H 0.19 111M111G 0.32 111 M111P 0.57 111 M111E 0.67 111 M111L 0.67 111 M111K 0.71111 M111T 0.76 111 M111F 0.78 111 M111D 0.79 111 M111V 0.93 112 S112Y0.01 112 S112R 0.01 112 S112P 0.01 112 S112H 0.38 112 S112V 0.48 112S112M 0.56 112 S112W 0.58 112 S112K 0.68 112 S112T 0.72 112 S112N 0.85112 S112F 0.88 112 S112A 0.94 113 V113S 0.57 113 V113G 0.58 113 V113K0.72 113 V113H 0.76 113 V113W 0.80 113 V113L 0.85 113 V113T 0.86 113V113D 0.87 113 V113E 0.94 113 V113C 0.94 113 V113F 0.96 113 V113Y 0.98114 L114H 0.01 114 L114E 0.01 114 L114Q 0.12 114 L114P 0.28 114 L114S0.55 114 L114V 0.60 114 L114N 0.77 115 V115I 0.99 116 T116Y 0.47 116T116V 0.57 116 T116R 0.62 116 T116L 0.68 116 T116W 0.75 116 T116I 0.76116 T116Q 0.77 116 T116P 0.84 116 T116G 0.90 116 T116E 0.91 116 T116A0.95 116 T116S 0.96 117 Q117W 0.71 117 Q117V 0.76 117 Q117G 0.79 117Q117S 0.87 118 V118K 0.01 118 V118W 0.01 118 V118E 0.01 118 V118R 0.07118 V118P 0.22 118 V118D 0.40 118 V118I 0.55 118 V118G 0.56 118 V118S0.82 118 V118A 0.85 118 V118T 0.92 118 V118M 0.93 118 V118F 1.00 119L119G 0.01 119 L119S 0.01 119 L119F 0.01 119 L119R 0.01 119 L119P 0.01119 L119T 0.10 119 L119N 0.11 119 L119V 0.15 119 L119W 0.20 119 L119C0.24 119 L119D 0.28 119 L119E 0.32 119 L119I 0.43 119 L119H 0.46 119L119Y 0.56 120 T120P 0.01 120 T120H 0.50 120 T120R 0.60 120 T120A 0.66120 T120Q 0.78 120 T120C 0.92 121 S121P 0.38 121 S121R 0.70 121 S121W0.77 121 S121K 0.78 121 S121G 0.99 122 A122G 0.01 122 A122D 0.06 122A122F 0.15 122 A122H 0.17 122 A122R 0.40 122 A122S 0.43 122 A122K 0.45122 A122E 0.47 122 A122T 0.52 122 A122P 0.55 122 A122I 0.65 122 A122N0.70 122 A122Q 0.74 122 A122W 0.86 122 A122V 0.89 122 A122M 0.94 123G123C 0.30 123 G123Q 0.31 123 G123T 0.54 123 G123E 0.56 123 G123V 0.59123 G123R 0.60 123 G123N 0.71 123 G123H 0.74 123 G123F 0.80 123 G123P0.81 123 G123D 0.84 124 G124I 0.01 124 G124H 0.01 124 G124M 0.01 124G124W 0.01 124 G124P 0.01 124 G124A 0.03 124 G124Q 0.21 124 G124T 0.32124 G124V 0.33 124 G124R 0.41 124 G124L 0.54 124 G124S 0.56 124 G124Y0.56 124 G124N 0.60 124 G124D 0.64 124 G124C 0.67 124 G124F 0.95 125V125W 0.25 125 V125E 0.39 125 V125R 0.47 125 V125C 0.54 125 V125D 0.54125 V125P 0.62 125 V125F 0.63 125 V125S 0.79 125 V125Y 0.81 125 V125A0.93 125 V125I 0.94 126 G126I 0.01 126 G126V 0.18 126 G126Y 0.23 126G126L 0.54 126 G126A 0.55 126 G126E 0.60 126 G126P 0.67 126 G126T 0.74126 G126R 0.76 126 G126N 0.85 126 G126S 0.90 126 G126C 0.98 127 T127L0.01 127 T127E 0.01 127 T127Q 0.15 127 T127I 0.20 127 T127H 0.60 127T127D 0.62 127 T127M 0.64 127 T127C 0.65 127 T127V 0.68 127 T127G 0.71127 T127P 0.77 127 T127S 0.83 128 T128D 0.66 129 Y129W 0.01 129 Y129G0.01 129 Y129K 0.01 129 Y129V 0.01 129 Y129T 0.14 129 Y129A 0.17 129Y129R 0.18 129 Y129M 0.21 129 Y129D 0.23 129 Y129L 0.27 129 Y129N 0.53129 Y129P 0.59 129 Y129C 0.61 129 Y129S 0.69 129 Y129F 0.71 130 P130T0.01 130 P130H 0.01 130 P130G 0.01 130 P130S 0.01 130 P130L 0.09 130P130E 0.22 130 P130W 0.28 130 P130V 0.37 130 P130I 0.41 130 P130A 0.44130 P130F 0.48 130 P130R 0.53 130 P130K 0.55 130 P130C 0.64 130 P130M0.76 131 A131W 0.01 131 A131D 0.40 131 A131Y 0.48 131 A131L 0.59 131A131S 0.68 131 A131P 0.71 131 A131Q 0.74 131 A131V 0.78 131 A131H 0.82131 A131G 0.87 131 A131E 0.97 132 P132V 0.01 132 P132T 0.01 132 P132W0.01 132 P132F 0.01 132 P132I 0.01 132 P132H 0.01 132 P132R 0.01 132P132D 0.01 133 K133C 0.01 133 K133A 0.10 133 K133V 0.23 133 K133G 0.31133 K133H 0.31 133 K133M 0.33 133 K133T 0.39 133 K133I 0.45 133 K133Q0.52 133 K133S 0.58 133 K133F 0.59 133 K133P 0.71 133 K133E 0.76 133K133R 0.83 133 K133W 0.99 134 V134Q 0.79 134 V134T 0.86 134 V134I 0.89135 L135T 0.01 135 L135W 0.01 135 L135K 0.01 135 L135S 0.01 135 L135F0.01 135 L135G 0.01 135 L135R 0.01 135 L135P 0.01 135 L135Q 0.17 135L135V 0.43 135 L135E 0.63 135 L135M 0.78 136 V136P 0.01 136 V136E 0.20136 V136N 0.40 137 V137N 0.01 137 V137G 0.26 137 V137S 0.29 137 V137I0.70 137 V137T 0.93 138 S138I 0.35 138 S138V 0.69 139 P139S 0.01 139P139G 0.01 139 P139R 0.01 139 P139C 0.01 139 P139D 0.01 139 P139E 0.01139 P139F 0.01 139 P139H 0.01 139 P139I 0.01 139 P139K 0.01 139 P139N0.01 139 P139Q 0.01 139 P139T 0.01 139 P139V 0.01 140 P140T 0.01 140P140S 0.01 140 P140V 0.01 140 P140W 0.01 140 P140I 0.01 140 P140Y 0.01140 P140Q 0.01 140 P140R 0.01 141 P141R 0.01 141 P141G 0.01 141 P141S0.02 141 P141T 0.12 141 P141V 0.16 141 P141Q 0.37 141 P141I 0.38 141P141L 0.65 141 P141H 0.79 141 P141N 0.97 142 L142W 0.01 142 L142I 0.28142 L142S 0.31 142 L142Q 0.33 142 L142V 0.33 142 L142P 0.44 142 L142F0.54 142 L142A 0.56 142 L142K 0.66 142 L142C 0.70 143 A143W 0.01 143A143P 0.39 143 A143G 0.42 143 A143S 0.63 143 A143F 0.68 143 A143Q 0.81143 A143N 0.82 143 A143T 0.97 143 A143R 0.99 143 A143V 0.99 144 P144G0.62 144 P144A 0.79 144 P144T 0.81 144 P144S 0.92 145 M145W 0.01 145M145G 0.26 145 M145E 0.48 145 M145I 0.53 145 M145Q 0.57 145 M145L 0.61145 M145V 0.63 145 M145R 0.69 145 M145F 0.77 145 M145P 0.78 145 M145S0.78 145 M145T 0.79 145 M145A 0.79 145 M145Y 0.82 145 M145C 0.93 146P146W 0.68 146 P146T 0.76 146 P146V 0.77 146 P146S 0.96 147 H147S 0.75147 H147T 0.84 147 H147I 0.92 147 H147V 0.92 147 H147R 0.94 147 H147A0.98 148 P148Q 0.98 149 W149R 0.01 149 W149E 0.01 149 W149P 0.01 149W149C 0.12 149 W149I 0.24 149 W149A 0.31 149 W149S 0.33 149 W149Q 0.40149 W149T 0.44 149 W149G 0.45 149 W149M 0.49 149 W149F 0.50 149 W149L0.64 149 W149Y 0.75 150 F150P 0.32 150 F150N 0.36 150 F150G 0.46 150F150V 0.51 150 F150A 0.54 150 F150T 0.58 150 F150W 0.62 150 F150M 0.63150 F150E 0.73 150 F150C 0.78 150 F150I 0.78 150 F150K 0.85 151 Q151L0.01 151 Q151V 0.01 151 Q151F 0.01 151 Q151I 0.01 151 Q151W 0.32 152L152I 0.61 152 L152P 0.61 152 L152T 0.69 152 L152Q 0.76 152 L152G 0.77152 L152S 0.84 152 L152D 0.86 152 L152V 0.88 152 L152R 0.91 152 L152K0.91 152 L152H 0.92 153 I153N 0.89 154 F154T 0.01 154 F154G 0.01 154F154V 0.01 154 F154S 0.29 154 F154Q 0.97 155 E155R 0.01 155 E155F 0.23155 E155V 0.47 155 E155I 0.65 155 E155Q 0.69 156 G156I 0.01 156 G156F0.73 156 G156W 0.90 156 G156L 0.94 156 G156V 0.97 157 G157R 0.01 157G157P 0.01 157 G157S 0.19 157 G157V 0.40 157 G157C 0.61 157 G157E 0.84157 G157M 0.85 157 G157A 0.87 157 G157D 0.94 157 G157T 0.99 158 E158V0.89 158 E158D 0.89 158 E158T 0.91 158 E158I 0.94 159 Q159A 0.28 159Q159C 0.31 159 Q159P 0.49 159 Q159D 0.63 159 Q159L 0.70 159 Q159G 0.72159 Q159S 0.73 159 Q159R 0.74 159 Q159M 0.84 159 Q159E 0.97 160 K160W0.01 160 K160G 0.30 160 K160H 0.57 160 K160S 0.70 160 K160L 0.95 160K160I 1.00 161 T161R 0.01 161 T161H 0.01 161 T161W 0.01 161 T161N 0.01161 T161G 0.43 161 T161C 0.56 161 T161S 0.57 161 T161I 0.98 163 E163F0.27 163 E163R 0.49 163 E163V 0.55 163 E163P 0.77 163 E163G 0.80 163E163H 0.82 163 E163S 0.85 163 E163W 0.98 164 L164Y 0.01 164 L164A 0.01164 L164D 0.01 164 L164E 0.01 164 L164G 0.01 164 L164H 0.12 164 L164F0.86 164 L164C 0.91 164 L164T 0.99 165 A165I 0.59 165 A165K 0.82 165A165Y 0.84 165 A165S 0.94 165 A165F 1.00 166 R166T 0.74 166 R166V 0.76166 R166G 0.91 166 R166S 0.95 168 Y168G 0.01 168 Y168T 0.01 168 Y168V0.01 168 Y168I 0.01 168 Y168C 0.01 168 Y168Q 0.01 169 S169P 0.89 169S169T 0.97 170 A170I 0.44 170 A170S 0.47 170 A170G 0.62 170 A170T 0.72170 A170V 0.74 170 A170K 0.83 170 A170W 0.83 170 A170L 0.85 170 A170Q0.89 170 A170Y 0.89 171 L171R 0.01 172 A172K 0.01 172 A172R 0.01 172A172E 0.01 172 A172Q 0.18 172 A172V 0.39 172 A172W 0.45 172 A172P 0.58172 A172I 0.58 172 A172T 0.71 172 A172N 0.76 172 A172G 0.84 172 A172S0.85 172 A172C 0.86 174 F174W 0.01 174 F174Q 0.46 174 F174C 0.48 174F174R 0.52 174 F174S 0.61 174 F174T 0.64 174 F174V 0.67 174 F174G 0.91175 M175P 0.08 175 M175A 0.66 175 M175Y 0.72 175 M175G 0.75 175 M175W0.76 175 M175V 0.81 175 M175Q 0.83 175 M175L 0.86 175 M175R 0.86 175M175T 0.90 176 K176S 0.72 176 K176G 0.73 176 K176P 0.78 176 K176L 0.92176 K176Y 0.93 176 K176N 0.94 176 K176T 0.97 176 K176Q 0.97 178 P178W0.02 179 F179Q 0.01 179 F179S 0.34 179 F179W 0.86 179 F179H 0.93 179F179N 0.95 180 F180K 0.01 180 F180T 0.01 180 F180R 0.01 180 F180S 0.01180 F180G 0.01 180 F180Q 0.01 181 D181Y 0.01 181 D181W 0.01 181 D181L0.01 181 D181T 0.01 181 D181V 0.01 181 D181R 0.22 181 D181K 0.47 181D181G 0.52 181 D181S 0.55 181 D181Q 0.60 181 D181P 0.66 181 D181E 0.72181 D181C 0.85 182 A182I 0.01 182 A182R 0.01 182 A182Q 0.01 182 A182P0.01 182 A182T 0.11 182 A182N 0.53 182 A182S 0.85 182 A182G 0.94 182A182C 0.99 183 G183S 0.01 183 G183Q 0.01 183 G183V 0.01 183 G183F 0.19183 G183H 0.95 183 G183D 0.99 184 S184T 0.60 184 S184H 0.74 184 S184G0.82 184 S184P 0.85 185 V185W 0.01 185 V185H 0.01 185 V185G 0.01 185V185D 0.01 185 V185S 0.53 185 V185Y 0.58 185 V185I 0.63 185 V185R 0.79185 V185K 0.79 185 V185C 0.83 185 V185E 0.88 185 V185T 0.91 185 V185L0.93 186 I186G 0.01 186 I186S 0.01 186 I186R 0.01 186 I186P 0.01 186I186T 0.23 186 I186V 0.48 186 I186F 0.76 187 S187P 0.01 187 S187T 0.23187 S187Q 0.35 187 S187W 0.52 187 S187R 0.55 187 S187V 0.58 187 S187F0.65 187 S187Y 0.80 188 T188H 0.01 188 T188R 0.01 188 T188F 0.01 188T188Y 0.09 188 T188I 0.10 188 T188V 0.15 188 T188L 0.42 188 T188M 0.75188 T188G 0.79 188 T188C 0.87 188 T188S 0.91 188 T188A 0.95 189 D189F0.37 189 D189R 0.39 189 D189N 0.57 189 D189V 0.71 189 D189W 0.76 189D189E 0.77 189 D189G 0.80 189 D189S 0.81 189 D189M 0.88 189 D189C 0.94189 D189H 0.95 189 D189P 0.97 190 G190V 0.01 190 G190S 0.01 190 G190Q0.29 190 G190W 0.41 190 G190R 0.51 190 G190K 0.57 190 G190L 0.82 191V191H 0.01 191 V191W 0.01 191 V191S 0.01 191 V191G 0.01 191 V191N 0.01191 V191I 0.02 192 D192S 0.01 192 D192P 0.01 192 D192F 0.01 192 D192H0.01 192 D192I 0.01 192 D192Q 0.01 192 D192R 0.01 192 D192T 0.01 192D192V 0.01 192 D192W 0.01 192 D192N 0.15 192 D192C 0.56 193 G193H 0.01193 G193C 0.01 193 G193T 0.01 193 G193N 0.01 194 I194S 0.01 194 I194A0.01 194 I194C 0.01 194 I194P 0.01 194 I194F 0.01 194 I194W 0.01 194I194R 0.01 194 I194Y 0.01 194 I194G 0.04 194 I194L 0.58 194 I194V 0.78195 H195S 0.08 195 H195C 0.10 195 H195L 0.18 195 H195N 0.22 195 H195R0.24 195 H195F 0.40 195 H195V 0.60 195 H195Q 0.96 195 H195A 0.98 196F196H 0.01 196 F196G 0.01 196 F196S 0.01 196 F196Q 0.01 196 F196W 0.38196 F196P 0.39 196 F196V 0.68 196 F196M 0.71 196 F196Y 0.97 197 T197R0.01 197 T197L 0.65 197 T197S 0.75 197 T197G 0.81 197 T197I 0.84 197T197C 0.86 197 T197V 0.89 197 T197N 0.91 199 A199M 0.93 199 A199S 0.99199 A199G 0.99 201 N201Y 0.01 201 N201T 0.01 201 N201V 0.01 201 N201R0.01 201 N201S 0.06 201 N201H 0.10 201 N201G 0.30 201 N201L 0.35 201N201F 0.67 201 N201E 0.72 203 D203V 0.50 203 D203W 0.52 203 D203E 0.90

The following Table provides variants that have protein performanceindices (“Prot. PI”) better than wild-type.

TABLE 10-8 Sites with Protein PI Values Better Than Wild-Type PosWT/Pos./Var. Prot. PI 2 A002Y 1.61 2 A002N 1.30 2 A002I 1.25 2 A002V1.18 2 A002T 1.17 2 A002S 1.15 5 I005M 1.29 7 C007A 1.22 7 C007G 1.07 7C007M 1.03 8 F008N 1.23 8 F008M 1.05 8 F008G 1.03 8 F008P 1.01 11 S011H1.06 11 S011A 1.04 11 S011D 1.03 11 S011E 1.01 11 S011Q 1.01 12 L012N1.06 12 L012Q 1.05 13 T013V 1.17 14 W014Y 1.02 16 W016Y 1.02 17 V017A1.21 17 V017E 1.11 17 V017F 1.09 17 V017I 1.08 17 V017K 1.06 17 V017T1.03 18 P018C 2.56 18 P018H 2.50 18 P018L 2.50 18 P018E 2.47 18 P018G2.47 18 P018N 2.35 18 P018V 2.30 18 P018Q 2.13 18 P018R 2.01 18 P018Y1.68 18 P018S 1.05 19 V019G 1.39 19 V019A 1.23 19 V019E 1.10 19 V019Q1.07 19 V019K 1.03 19 V019M 1.00 20 E020G 1.11 20 E020P 1.08 20 E020A1.08 20 E020N 1.01 20 E020V 1.01 22 G022A 1.07 22 G022I 1.03 23 A023F1.03 24 P024T 1.43 24 P024G 1.34 24 P024S 1.31 24 P024H 1.15 24 P024I1.11 24 P024L 1.06 25 T025C 1.37 25 T025V 1.30 25 T025G 1.27 25 T025A1.23 25 T025I 1.19 25 T025P 1.10 25 T025M 1.04 29 A029G 1.22 29 A029P1.07 29 A029M 1.06 29 A029D 1.06 29 A029V 1.05 29 A029S 1.05 29 A029T1.02 29 A029E 1.02 30 P030E 1.20 30 P030A 1.15 30 P030S 1.12 30 P030L1.07 30 P030Q 1.06 30 P030K 1.06 30 P030H 1.05 30 P030Y 1.04 32 V032M1.11 32 V032A 1.10 32 V032I 1.08 32 V032Q 1.03 32 V032L 1.01 35 T035C1.16 36 G036C 1.09 36 G036N 1.08 36 G036Q 1.07 36 G036S 1.06 36 G036A1.00 37 V037N 1.09 39 A039V 1.18 39 A039E 1.03 46 F046A 1.05 46 F046C1.01 47 E047I 1.02 54 S054A 1.33 54 S054C 1.21 54 S054E 1.16 54 S054D1.08 54 S054H 1.06 54 S054N 1.01 54 S054M 1.01 55 A055N 1.12 55 A055S1.08 56 R056Q 1.02 58 T058V 1.13 60 I060A 1.20 60 I060M 1.14 60 I060V1.06 60 I060L 1.02 61 D061A 1.41 61 D061N 1.12 61 D061V 1.10 61 D061Y1.03 61 D061Q 1.02 61 D061L 1.00 62 D062A 1.06 62 D062M 1.06 63 P063S1.17 63 P063Y 1.12 63 P063M 1.09 63 P063Q 1.08 63 P063A 1.06 63 P063V1.06 63 P063R 1.02 63 P063T 1.02 64 T064Q 1.13 64 T064M 1.07 64 T064R1.05 64 T064C 1.05 64 T064S 1.03 66 P066Q 1.91 66 P066G 1.78 66 P066N1.62 66 P066C 1.51 66 P066I 1.51 66 P066R 1.26 66 P066H 1.23 66 P066V1.12 66 P066Y 1.08 66 P066A 1.03 66 P066F 1.02 67 R067Q 1.60 67 R067L1.46 67 R067A 1.39 67 R067V 1.24 67 R067P 1.04 67 R067F 1.01 68 L068A1.07 68 L068V 1.01 68 L068G 1.00 69 N069C 1.18 69 N069G 1.06 69 N069D1.05 69 N069S 1.03 70 G070A 1.08 72 S072L 1.07 72 S072A 1.06 72 S072Y1.03 73 Y073N 1.25 73 Y073Q 1.20 73 Y073C 1.18 73 Y073D 1.09 73 Y073V1.08 73 Y073M 1.05 73 Y073L 1.03 74 L074I 1.45 74 L074Y 1.19 74 L074V1.18 74 L074A 1.01 75 P075M 1.22 75 P075S 1.18 75 P075T 1.10 75 P075Y1.08 75 P075C 1.06 75 P075Q 1.04 75 P075L 1.02 75 P075E 1.00 76 S076W1.06 77 C077L 1.44 77 C077V 1.33 77 C077A 1.20 77 C077S 1.19 77 C077T1.18 78 L078I 1.06 78 L078V 1.04 79 A079C 1.16 79 A079E 1.12 79 A079S1.09 79 A079Q 1.05 79 A079M 1.04 79 A079R 1.02 80 T080S 1.12 80 T080E1.02 80 T080Q 1.02 82 L082G 1.24 82 L082R 1.15 82 L082V 1.14 82 L082S1.13 82 L082P 1.11 82 L082M 1.07 82 L082K 1.03 82 L082A 1.00 83 P083G1.01 84 L084V 1.23 86 L086Q 3.66 89 I089V 1.09 89 I089L 1.07 93 T093Q2.03 96 T096A 1.32 96 T096V 1.12 96 T096S 1.05 96 T096G 1.03 97 K097A1.11 97 K097R 1.02 98 A098S 1.17 98 A098T 1.03 98 A098N 1.01 99 Y099S1.45 99 Y099L 1.39 99 Y099H 1.30 99 Y099A 1.29 99 Y099V 1.28 99 Y099G1.23 99 Y099W 1.20 99 Y099I 1.11 100 F100M 1.20 100 F100N 1.12 100 F100W1.06 100 F100S 1.02 101 R101L 1.33 101 R101N 1.11 101 R101Q 1.03 101R101D 1.02 102 R102Q 1.09 103 T103G 1.20 103 T103S 1.14 103 T103H 1.14103 T103N 1.07 103 T103K 1.05 103 T103P 1.01 104 P104S 1.44 104 P104V1.40 104 P104E 1.37 104 P104C 1.34 104 P104N 1.32 104 P104T 1.29 104P104G 1.25 104 P104Q 1.24 104 P104H 1.11 104 P104I 1.07 104 P104M 1.01105 L105Y 1.18 105 L105H 1.07 105 L105G 1.07 105 L105C 1.05 105 L105Q1.03 105 L105T 1.00 105 L105P 1.00 106 D106E 1.02 107 I107S 1.05 107I107V 1.04 107 I107C 1.00 108 A108G 1.15 108 A108S 1.14 108 A108T 1.08109 L109E 1.24 109 L1091 1.21 109 L109D 1.15 109 L109N 1.13 109 L109F1.11 109 L109Q 1.08 109 L109A 1.07 109 L109H 1.06 109 L109V 1.06 109L109M 1.00 110 G110S 1.01 112 S112N 1.09 112 S112E 1.05 113 V113C 1.06113 V113N 1.01 114 L114C 1.10 114 L114A 1.03 114 L114M 1.00 115 V115I1.14 115 V115C 1.14 115 V115A 1.11 115 V115M 1.05 115 V115L 1.02 116T116N 1.68 116 T116H 1.48 116 T116G 1.44 116 T116C 1.30 116 T116E 1.29116 T116Q 1.29 116 T116M 1.28 116 T116S 1.24 116 T116Y 1.09 116 T116A1.08 116 T116R 1.03 116 T116L 1.03 117 Q117S 1.13 117 Q117H 1.12 117Q117E 1.10 117 Q117T 1.06 117 Q117A 1.03 118 V118C 1.28 118 V118A 1.20118 V118I 1.01 119 L119C 1.18 119 L119A 1.18 119 L119N 1.14 119 L119I1.06 119 L119S 1.05 119 L119V 1.04 119 L119E 1.04 119 L119R 1.00 120T120S 1.35 120 T120E 1.19 120 T120C 1.14 120 T120K 1.12 120 T120N 1.10120 T120A 1.09 120 T120H 1.07 120 T120Q 1.05 120 T120Y 1.01 120 T120L1.00 121 S121N 1.17 121 S121L 1.12 121 S121A 1.10 121 S121C 1.09 121S121G 1.07 121 S121R 1.06 121 S121K 1.04 121 S121E 1.01 121 S121Q 1.01122 A122N 1.11 122 A122L 1.07 122 A122P 1.07 122 A122M 1.06 122 A122V1.05 122 A122S 1.05 122 A122E 1.04 122 A122I 1.04 122 A122Q 1.02 124G124M 1.36 124 G124A 1.20 124 G124N 1.18 124 G124C 1.07 124 G124Q 1.02125 V125I 1.05 126 G126N 1.04 126 G126E 1.02 126 G126A 1.02 127 T127A1.10 127 T127S 1.08 127 T127V 1.06 127 T127C 1.04 127 T127G 1.04 127T127D 1.03 127 T127E 1.03 127 T127M 1.02 128 T128N 1.29 128 T128M 1.28128 T128Q 1.24 128 T128A 1.23 128 T128H 1.19 128 T128P 1.18 128 T128D1.14 128 T128K 1.10 128 T128S 1.07 128 T128V 1.05 128 T128R 1.03 128T128F 1.01 129 Y129F 1.44 129 Y129C 1.42 129 Y129A 1.39 129 Y129D 1.35129 Y129M 1.28 129 Y129N 1.24 129 Y129L 1.22 129 Y129P 1.11 129 Y129G1.10 129 Y129S 1.08 129 Y129W 1.01 129 Y129V 1.00 130 P130G 1.11 130P130E 1.08 130 P130K 1.05 130 P130A 1.03 130 P130M 1.03 133 K133Q 1.13133 K133S 1.02 133 K133A 1.01 133 K133R 1.01 133 K133E 1.01 135 L135M1.01 136 V136L 1.03 138 S138A 1.44 138 S138C 1.17 138 S138G 1.09 141P141A 1.13 141 P141G 1.02 142 L142I 1.05 143 A143G 1.17 145 M145I 1.16145 M145L 1.07 147 H147L 1.09 147 H147C 1.04 149 W149G 1.39 149 W149A1.35 149 W149M 1.32 149 W149S 1.28 149 W149F 1.27 149 W149Y 1.15 149W149Q 1.10 149 W149L 1.06 150 F150A 1.70 150 F150M 1.69 150 F150N 1.52150 F150C 1.41 150 F150P 1.38 150 F150K 1.33 150 F150E 1.32 150 F150T1.27 150 F150V 1.26 150 F150W 1.26 150 F150Y 1.24 150 F150I 1.19 150F150L 1.14 150 F150G 1.13 150 F150H 1.09 151 Q151K 1.04 153 I153N 1.04157 G157A 1.00 159 Q159E 1.14 159 Q159A 1.13 159 Q159G 1.03 161 T161C1.01 162 T162C 1.17 162 T162I 1.16 162 T162H 1.08 162 T162L 1.05 162T162F 1.05 162 T162Y 1.03 164 L164M 1.09 164 L164V 1.08 165 A165G 1.14165 A165Q 1.05 165 A165S 1.05 166 R166M 1.26 166 R166K 1.19 166 R166G1.19 166 R166N 1.16 166 R166D 1.16 166 R166A 1.12 166 R166L 1.08 166R166T 1.04 167 V167L 1.13 167 V167H 1.12 167 V167G 1.08 167 V167M 1.04167 V167I 1.04 167 V167S 1.04 167 V167C 1.01 168 Y168F 1.28 168 Y168L1.27 170 A170C 1.02 171 L171I 1.16 172 A172C 1.09 172 A172G 1.07 175M175Y 1.35 175 M175L 1.19 175 M175W 1.14 175 M175N 1.11 175 M175R 1.02176 K176R 1.06 176 K176Q 1.02 178 P178E 1.05 182 A182C 1.03 183 G183S1.08 184 S184E 1.39 184 S184A 1.31 184 S184M 1.25 184 S184G 1.15 184S184D 1.15 184 S184C 1.14 184 S184Q 1.09 184 S184H 1.07 184 S184N 1.03184 S184V 1.03 184 S184K 1.02 185 V185I 1.03 186 I186M 1.11 188 T188C2.04 188 T188I 1.85 188 T188L 1.76 188 T188M 1.60 188 T188V 1.53 188T188S 1.52 188 T188R 1.41 188 T188A 1.40 188 T188G 1.32 188 T188N 1.24191 V191C 1.04 194 I194L 1.32 194 I194C 1.17 194 I194A 1.15 194 I194W1.12 194 I194V 1.03 194 I194Y 1.01 196 F196L 1.09 201 N201H 1.49

The following Table provides variants that have a PAD PI that is greaterthan 1.5, a PAF that is greater than or equal to 0.1, and a protein PIthat is greater than or equal to 0.1

TABLE 10-9 PAD PI > 1.5 with PAF ≧ 0.1 and protein PI ≧ 0.1 Wild-TypeVariant Amino Acid/Pos. Amino Acid M1 L K3 A, C, H, I, L R4 A I5 A, C,E, L L6 A C7 K T13 A, C P18 C, E, G, H, L, Q, R, V, Y E20 C, Q D21 A, G,K, L, Y G22 A P24 L E26 L R27 A, K, L F28 D, L P30 T, V D31 L, N V32 A,D, E, G, I, K, L, M, N, Q, W R33 C, G, K, L T35 A, C, I, M G36 K Q40 D,G, K, S, T, W, Y Q41 A, K, L G43 E, L A44 C F46 L V48 A, C, L, M, P I49A E51 A L53 H N59 A, C, D, E, F, G, K, L, Q, S, T, V, W, Y D61 I, K, RN69 H, I, K, V S72 A, C, G, H, M, N P75 D, G, K, S, T, W, Y S76 D, E, G,M T80 G H81 M P83 A, M D85 F, G L86 C V87 C, L I89 A T96 A, C, L, M A98D F100 A, M R102 A, L P104 C, E, I, M L105 C, F, W D106 V I107 T G110 E,L V115 G Q117 A, M V118 Q T120 E, I, Y S121 A, C, V T128 F, K, L, R, YP132 A, C, E, G, L, Q, S, Y K133 L V134 A, M V136 A P140 A P144 H, YP146 C, F, H, L P148 F Q151 A, C, D, E, H, K, P, R, S, T, Y L152 W I153F, H, K, P, S, T F154 Y E155 A, L, M, N, P, Y G156 D, M, T G157 H E158F, K, L, M, N, Y T161 M, Q T162 C, F, G, H, I, K, L, M, N, P, Q, S, W, YE163 A, L, Y A165 D, L, M R166 A, D, H, L V167 A, C, D, G, H, L, M, P,Q, R, S, T, Y Y168 F, L S169 I L171 A, C, F, K, M, N, Q, S S173 A, C, E,F, I, K, L, M, P, R, V, W, Y F174 A, L, M, Y P178 A, D, E, G, K, L, M,Q, S, T, V, Y F179 L G190 A, H, M V191 A, C, D, E, F, K, L, M, Q, R, YG193 S, V T197 M E198 C, L, M, N, P, R, W, Y A199 C, K, L, Y R202 A, C,E, F, G, H, I, L, M, S, T, W D203 A, C, H, L, R G205 A V206 C, E, F, G,H, K, L, M, N, P, R A209 E, L E210 D, K Q211 M, N, P S214 A, C, D, F, G,I, K, L, R, T, V, W, L215 E, M, T, V, Y

The following Table provides variants with a PAD PI that is less than0.5, a PAF that is greater than or equal to 0.1, and a protein PI thatis greater than or equal to 0.1.

TABLE 10-10 PAD PI < 0.5 with PAF ≧ 0.1, and Protein PI ≧ 0.1 Wild-TypeAmino Acid Residue/Pos. Variant(s) A2 Y R4 I, L, V I5 S L6 S, T, V F8 RD10 G L12 A, C, F, G, K, Q, R, S, T, V W14 F, G, I, K, L, R, S, T, V G15C, N P18 S V19 M, Q, R G22 K, W A23 G, R, S, T25 G, H, I, K, L, M, P, R,W E26 N, S, T, W R27 P, T, W F28 G A29 T, V T35 N, Q, V G36 S, T L38 G,S Q41 S, V L42 Q, S, T G43 P, Q, S, V D45 R, S, T F46 T E47 P V48 S I49P, R E50 V E51 I, V G52 H, L, S, V L53 E, G, K, R, S S54 F, G, I, K, L,R, T, V, W, Y A55 I, R, T, V R56 C, G, S, T T57 C, N T58 A, M N59 M, RI60 P D62 C, G, H, I, L, R, S, T, V, W T64 R D65 H, R, S, V, Y P66 G, N,Q R67 E, F, G, L, N, P, Q, T, V, W L68 A, C, E, F, G, H, M, N, P, Q, R,S, T, Y N69 Y G70 C, T S72 W, Y Y73 L, R P75 M, R S76 F, W, Y C77 F, W,Y L78 M A79 C, E, H, M, N, Q, R T80 H, I, K, L, W, Y H81 R, Y L82 G, H,R, S, T, V, W P83 T, V L84 A, T, V, W D85 I, L, V, W L86 H, S, T, V, WV87 A, F, G, S, T, Y I88 T, V I89 S M90 S, T, V L91 T, V T93 S, Y N94 H,L, T, V T96 I, R, W, Y K97 G, I, L, P, Q, S, T, V, Y A98 T Y99 S, V F100E, K, W R101 K, Q, V, W R102 C, G T103 A, C, F, G, H, I, K, L, N, P, R,S, V, W, Y P104 R, T L105 V I107 P, Q L109 A, D, E, F, H, I, Q, R, S, WG110 Q, S, T M111 G, H, R, S S112 H, R, V, Y L114 Q T116 Y V118 P, R, WL119 C, D, E, F, G, H, I, N, R, S, T, V, W T120 H S121 P A122 D, E, F,G, H, K, R, S G123 C G124 A, H, I, M, Q, R, T, V, W V125 E, R, W G126 I,V, Y T127 E, I, L, Q Y129 A, D, G, K, L, M, R, T, V, W P130 A, E, F, G,H, I, L, S, T, V, W A131 D, W, Y P132 F, H, I, T, V K133 A, C, G, H, I,M, T, V L135 F, Q, S, T, V V137 S S138 I P139 S P140 S P141 G, I, Q, R,S, T, V L142 Q, S, V A143 G, P, W M145 E, G, W W149 A, C, F, G, I, M, Q,S, T F150 G, N, P, W E155 F, R, V G156 I G157 R, S, V Q159 A, C, P K160G T161 G, H, R, W E163 F, R Y168 C, I, V A170 I, S A172 Q, V F174 C, Q,W F179 Q, S G190 S, V, W V191 G, H, I, N, S, W G193 C, H, T I194 A, C,G, S F196 G, Q, W T197 R N201 G, H, L, R, S, T, V, Y D203 V L208 Q, S,V, Y V212 G L215 A, C, G, K, P, R L216 G, I, T

In addition to the assay results described above, various mutations werefound to result in unstable protein such that perhydrolase protein wasnot expressed. Thus, in contrast to the substitutions that resulted inenhanced expression as compared to wild-type, there were somesubstitutions that are not as favorable, at least under the conditionsused herein. However, it is not intended that the present inventionexclude these substitutions, as it is contemplated that thesesubstitutions, taken alone or in combination will find use inalternative embodiments of the present invention.

TABLE 10-11 Mutations that Produced Unstable Protein Wild-Type/ VariantPos. Amino Acid M1 A, E, F, G, K, N, P, R, S, T, W I5 W C7 L, P, T, W G9A, C, E, K, L, P, Q, R, V T13 F, R, W G15 H, K, L, R, Y P18 A D21 V F28H, I, R R33 D, E, H, P, W W34 K T35 K, L, P, W, Y G36 P V37 Q, R L38 WA39 F L42 D A44 D, H, P F46 H V48 W E51 P R56 H, K, P, W, Y T57 W T58 E,G, K, P, R, W, Y L74 D, H, P, Q, R, T C77 N, P L78 A, P, R, S A79 V L86F I88 R, Y I89 D, R L91 H, K, P, R, W, Y G92 A, D, L, M, P, R, T, W, YT93 P, R, V, W D95 A, D, G, H, K, L, N, Q, R, S, T, V, W, Y K97 D P104A, L L105 A, M I107 H, W A108 D, F, H, I, N, P, R G110 L L114 F, K, R,W, Y V115 H, K, V134 D, K, R, W, Y V136 R, W V137 D, E, F, P, R, W S138E, F, H, L, M, Q, R, W, Y P139 L, W, Y P140 D, K, L, M L142 D, G, M, N,R, T H147 G F154 E, L, P, T161 D, E, P Y168 D, E, H, K, N, P, R, S, WL171 D F179 A, P, R F180 E D181 F, H, I, M, N A182 H, K, L, M, W, Y I186K, W, Y T188 D, K, P, Q, W F196 A, K, N, R

The following Table provides performance indices obtained in PAF and PADassays for various variants, as well as the protein performance index.

TABLE 10-12 Performance Indices Wild-Type Res./Pos. Mut. PAF PI PAD PIProt. PI M1 A −0.12 −0.12 −0.01 M1 E −0.12 −0.12 −0.01 M1 F −0.12 −0.12−0.01 M1 G −0.12 −0.12 −0.01 M1 I 0.96 1.19 0.31 M1 K −0.12 −0.12 −0.01M1 L 0.75 2.11 0.30 M1 M 1.00 1.00 1.00 M1 N −0.12 −0.12 −0.01 M1 P−0.12 −0.12 −0.01 M1 R −0.12 −0.12 −0.01 M1 S −0.12 −0.12 −0.01 M1 T−0.12 −0.12 −0.01 M1 V 0.87 0.94 0.52 M1 W −0.12 −0.12 −0.01 A2 A 1.001.00 1.00 A2 D 1.30 1.05 0.77 A2 E 0.61 1.38 0.52 A2 F 1.24 0.93 0.89 A2G 1.15 0.84 0.95 A2 I 1.18 0.61 1.25 A2 N 0.93 0.59 1.30 A2 P 0.52 1.170.68 A2 Q 0.81 1.29 0.65 A2 R 0.90 1.17 0.70 A2 S 1.01 0.66 1.15 A2 T0.98 0.61 1.17 A2 V 0.89 0.60 1.18 A2 W 1.75 1.17 0.53 A2 Y 0.84 0.461.61 K3 A 0.86 2.14 0.48 K3 C 0.81 1.52 0.67 K3 E 0.12 3.51 0.11 K3 G0.72 3.74 0.08 K3 H 1.01 1.89 0.30 K3 I 1.05 2.44 0.16 K3 K 1.00 1.001.00 K3 L 1.04 1.84 0.50 K3 M 0.85 1.44 0.71 K3 P 0.80 1.45 0.59 K3 Q0.87 1.19 0.69 K3 R 0.87 1.29 0.46 K3 S 0.94 1.17 0.44 K3 T 1.01 1.030.71 K3 V 0.81 0.84 0.33 K3 Y 1.06 1.39 0.86 R4 A 0.41 1.64 0.29 R4 C0.71 1.34 0.35 R4 D 0.27 1.18 0.32 R4 E 0.32 0.97 0.25 R4 G 0.79 0.790.41 R4 H 0.92 0.99 0.59 R4 I 0.24 0.15 0.18 R4 L 0.21 −0.03 0.18 R4 P0.14 1.44 0.13 R4 Q 1.03 0.99 0.70 R4 R 1.00 1.00 1.00 R4 S 0.65 0.910.64 R4 T 0.80 1.00 0.69 R4 V 0.29 0.08 0.22 R4 W 0.04 0.48 0.12 R4 Y0.63 0.98 0.39 I5 A 0.60 1.88 0.62 I5 C 0.44 2.47 0.54 I5 D −0.13 3.110.06 I5 E 0.67 1.59 0.33 I5 F −0.13 0.15 0.06 I5 G 0.05 −3.88 0.10 I5 H0.55 0.63 0.18 I5 I 1.00 1.00 1.00 I5 L 0.80 1.63 0.96 I5 M 0.63 1.091.29 I5 N −0.13 −2.15 0.12 I5 P −0.13 −0.86 0.08 I5 R −0.13 −6.48 0.08I5 S 1.02 0.37 0.39 I5 T 1.12 0.72 0.25 I5 V 0.94 0.92 0.54 I5 W −0.13−0.44 −0.01 L6 A 0.87 1.99 0.26 L6 C 0.85 1.22 0.55 L6 E −0.20 −0.590.09 L6 G 0.23 −3.45 0.12 L6 H 0.23 −1.08 0.09 L6 I 1.07 0.82 0.86 L6 K0.41 −1.16 0.05 L6 L 1.00 1.00 1.00 L6 M 0.92 1.44 0.63 L6 Q −0.20 −1.630.12 L6 R 0.06 −1.59 0.12 L6 S 0.58 −1.26 0.23 L6 T 1.06 0.35 0.40 L6 V1.07 0.35 0.44 L6 W 0.06 −2.97 0.09 C7 A 1.42 1.03 1.22 C7 C 1.00 1.001.00 C7 E −0.26 1.63 0.20 C7 G 1.39 0.69 1.07 C7 H 1.73 1.37 0.41 C7 I1.76 1.48 0.31 C7 K 2.69 2.95 0.21 C7 L −0.26 −0.16 −0.01 C7 M 1.13 0.681.03 C7 P −0.26 −0.16 −0.01 C7 R 0.22 −1.04 0.15 C7 S 0.62 −2.83 0.10 C7T −0.26 −0.16 −0.01 C7 W −0.26 −0.16 −0.01 C7 Y 2.09 0.54 0.67 F8 A 0.551.33 0.96 F8 C −0.11 4.01 0.10 F8 F 1.00 1.00 1.00 F8 G 1.09 0.65 1.03F8 H 1.02 0.64 0.97 F8 K 0.81 0.83 0.95 F8 L 0.77 1.31 0.90 F8 M 0.561.11 1.05 F8 N −0.11 0.96 1.23 F8 P 1.00 0.83 1.01 F8 R 1.43 0.46 0.73F8 S 0.71 −2.75 0.13 F8 T 0.88 0.77 0.94 F8 V 1.18 0.85 0.88 F8 Y 0.960.90 0.85 G9 A −0.15 −0.18 −0.01 G9 C −0.15 −0.18 −0.01 G9 E −0.15 −0.18−0.01 G9 G 1.00 1.00 1.00 G9 H 0.29 −0.06 0.16 G9 K −0.15 −0.18 −0.01 G9L −0.15 −0.18 −0.01 G9 P −0.15 −0.18 −0.01 G9 Q −0.15 −0.18 −0.01 G9 R−0.15 −0.18 −0.01 G9 T 0.21 −2.56 0.12 G9 V −0.15 −0.18 −0.01 D10 A−0.29 −14.24 0.02 D10 D 1.00 1.00 1.00 D10 E 0.01 0.15 0.72 D10 G 0.41−0.92 0.17 D10 I 1.28 −6.86 0.04 D10 K 2.13 −5.30 0.02 D10 L 3.97 2.040.02 D10 M −0.29 −5.94 0.04 D10 N −0.29 −2.23 0.07 D10 P −0.29 −4.160.05 D10 R 0.22 −4.36 0.06 D10 S 0.79 −0.58 0.06 D10 T 1.47 −0.45 0.06D10 V 0.98 −4.22 0.06 D10 W 3.18 −3.70 0.02 D10 Y 1.51 −4.97 0.03 S11 A0.25 0.53 1.04 S11 D −0.25 −0.22 1.03 S11 E −0.25 −0.23 1.01 S11 F −0.25−0.13 0.68 S11 G −0.25 −0.09 0.86 S11 H −0.25 0.33 1.06 S11 I −0.25 0.560.63 S11 K −0.25 0.40 0.62 S11 L −0.25 −0.22 0.68 S11 Q −0.25 −0.26 1.01S11 R −0.25 −0.08 0.69 S11 S 1.00 1.00 1.00 S11 T 0.04 −0.36 0.87 S11 V0.03 −0.15 0.59 L12 A 1.10 0.07 0.71 L12 C 2.29 0.22 0.81 L12 D 0.040.00 0.39 L12 F 0.13 0.17 0.60 L12 G 0.44 −0.06 0.60 L12 H 0.02 0.160.77 L12 K 0.18 0.13 0.40 L12 L 1.00 1.00 1.00 L12 N 0.53 0.66 1.06 L12P 0.03 −0.16 0.31 L12 Q 2.65 0.22 1.05 L12 R 0.23 −0.02 0.34 L12 S 0.54−0.07 0.80 L12 T 0.68 0.06 0.89 L12 V 0.98 −0.05 0.51 L12 W 0.03 0.020.33 T13 A 0.25 1.88 0.72 T13 C 0.56 1.55 0.78 T13 E −0.10 1.09 0.44 T13F −0.10 −0.11 −0.02 T13 G 0.32 0.77 0.57 T13 I 0.12 1.05 0.69 T13 L 0.551.47 0.76 T13 M 0.17 1.47 0.94 T13 N −0.10 2.61 0.27 T13 P −0.10 2.730.17 T13 Q 0.01 0.51 0.98 T13 R −0.10 −0.11 −0.02 T13 S 0.73 0.68 0.88T13 T 1.00 1.00 1.00 T13 V 0.19 0.63 1.17 T13 W −0.10 −0.11 −0.02 W14 A−0.23 0.27 0.94 W14 E 0.06 0.15 0.80 W14 F 0.29 0.22 0.71 W14 G 0.30−0.97 0.70 W14 I 0.33 −0.42 0.66 W14 K 0.29 −0.17 0.71 W14 L 0.25 −0.360.82 W14 N −0.23 −0.12 0.81 W14 P −0.23 −0.29 0.34 W14 R 0.23 −0.40 0.66W14 S 0.31 −0.99 0.69 W14 T 0.24 −0.77 0.64 W14 V 0.26 −0.49 0.58 W14 W1.00 1.00 1.00 W14 Y 0.31 0.66 1.02 G15 A 1.54 0.61 0.87 G15 C 0.71−0.27 0.66 G15 D −0.18 0.01 0.26 G15 E −0.18 −1.42 0.11 G15 G 1.00 1.001.00 G15 H −0.18 −0.14 −0.01 G15 K −0.18 −0.14 −0.01 G15 L −0.18 −0.14−0.01 G15 N 0.46 −0.63 0.71 G15 P −0.18 −5.42 0.09 G15 R −0.18 −0.14−0.01 G15 S 1.05 0.63 0.76 G15 Y −0.18 −0.14 −0.01 W16 A 0.12 0.55 0.50W16 D 0.02 0.57 0.32 W16 E 0.06 0.65 0.46 W16 G 0.05 −0.07 0.38 W16 H0.03 −0.02 0.55 W16 I 0.02 1.06 0.74 W16 K 0.01 1.03 0.73 W16 L −0.481.16 0.76 W16 M 0.04 0.37 0.56 W16 N 0.02 −0.03 0.43 W16 P 0.03 0.150.37 W16 Q 0.05 0.31 0.47 W16 R 0.03 −0.41 0.30 W16 S 0.09 −0.17 0.39W16 T 0.03 −0.31 0.41 W16 V 0.01 0.88 0.76 W16 W 1.00 1.00 1.00 W16 Y0.22 1.09 1.02 V17 A 1.01 0.68 1.21 V17 E 0.82 0.75 1.11 V17 F 0.92 0.851.09 V17 G 1.17 0.84 0.93 V17 I 0.95 0.99 1.08 V17 K 0.94 0.84 1.06 V17L 0.90 1.00 0.76 V17 P 0.77 0.96 0.97 V17 R 1.10 0.94 0.76 V17 S 0.961.04 0.89 V17 T 0.93 0.86 1.03 V17 V 1.00 1.00 1.00 V17 Y 0.91 0.88 0.99P18 A −0.28 −0.94 −0.03 P18 C 1.26 4.16 2.56 P18 E 1.22 4.87 2.47 P18 G1.07 4.96 2.47 P18 H 1.12 6.05 2.50 P18 L 0.93 7.40 2.50 P18 N 1.33 1.422.35 P18 P 1.00 1.00 1.00 P18 Q 1.12 3.26 2.13 P18 R 1.16 3.97 2.01 P18S 0.11 0.07 1.05 P18 V 1.19 4.85 2.30 P18 Y 1.33 4.17 1.68 V19 A 0.610.55 1.23 V19 D 0.77 0.79 0.80 V19 E 0.74 0.62 1.10 V19 G 1.32 0.56 1.39V19 K 0.96 0.97 1.03 V19 L 1.00 0.91 0.90 V19 M 0.33 0.12 1.00 V19 P0.00 −0.41 0.76 V19 Q 0.93 0.40 1.07 V19 R 1.03 0.34 0.82 V19 S 1.240.57 0.80 V19 V 1.00 1.00 1.00 V19 Y 0.94 0.70 0.92 E20 A 1.29 1.28 1.08E20 C 1.57 1.76 0.71 E20 D 0.87 1.14 0.97 E20 E 1.00 1.00 1.00 E20 G2.36 0.78 1.11 E20 H 2.17 1.20 0.92 E20 L 2.20 0.73 0.92 E20 N 1.40 1.341.01 E20 P 1.00 1.43 1.08 E20 Q 1.27 1.56 0.99 E20 S 2.01 1.18 0.91 E20T 2.22 1.25 0.94 E20 V 2.11 1.27 1.01 E20 W 2.94 1.30 0.79 D21 A 1.461.75 0.84 D21 D 1.00 1.00 1.00 D21 E 0.84 1.39 0.85 D21 F 1.30 1.41 0.81D21 G 1.37 1.76 0.93 D21 K 1.58 1.80 0.74 D21 L 1.46 1.57 0.82 D21 P0.81 0.86 0.74 D21 S 1.24 1.11 0.73 D21 V −0.17 −0.12 −0.02 D21 W 1.551.44 0.61 D21 Y 1.30 2.01 0.42 G22 A 1.55 1.66 1.07 G22 E 0.15 1.19 0.56G22 G 1.00 1.00 1.00 G22 I 0.37 1.03 1.03 G22 K 0.23 −0.22 0.78 G22 L0.38 1.35 0.84 G22 P 0.28 1.36 0.80 G22 Q 0.35 1.44 0.96 G22 R 0.11 0.560.73 G22 S 1.02 0.98 0.94 G22 T 1.03 1.16 0.80 G22 V 0.40 0.85 0.89 G22W 0.25 0.23 0.58 A23 A 1.00 1.00 1.00 A23 F 0.05 0.44 1.03 A23 G 0.450.35 0.93 A23 H 0.16 1.04 0.93 A23 L 0.30 1.30 0.75 A23 M 0.85 0.95 0.90A23 P −0.11 0.73 0.82 A23 Q 0.23 0.73 0.91 A23 R 0.11 0.28 0.80 A23 S0.69 0.34 0.87 A23 V 0.20 0.60 0.73 A23 W 0.29 0.80 0.71 A23 Y 0.20 0.960.73 P24 A 0.54 0.68 0.88 P24 C 0.54 1.04 0.87 P24 G 0.49 0.76 1.34 P24H 0.42 0.97 1.15 P24 I 0.42 0.85 1.11 P24 K 0.52 1.36 0.71 P24 L 0.581.51 1.06 P24 P 1.00 1.00 1.00 P24 Q 0.50 0.65 0.93 P24 R 0.58 0.91 0.85P24 S 0.53 0.61 1.31 P24 T 0.44 0.66 1.43 T25 A 1.33 0.86 1.23 T25 C0.67 0.51 1.37 T25 D 0.03 −0.07 0.87 T25 E 0.08 −0.29 0.98 T25 G 1.860.43 1.27 T25 H 0.42 −0.02 0.94 T25 I 1.02 0.35 1.19 T25 K 0.36 0.130.87 T25 L 0.40 −0.04 0.95 T25 M 0.29 −0.10 1.04 T25 P 0.97 −0.05 1.10T25 R 0.32 −0.06 0.94 T25 S 1.60 0.58 0.95 T25 T 1.00 1.00 1.00 T25 V0.91 0.51 1.30 T25 W 0.33 0.14 0.86 E26 A 1.93 1.45 0.79 E26 C 1.40 0.940.82 E26 D 0.65 1.39 0.90 E26 E 1.00 1.00 1.00 E26 G 1.28 0.87 0.82 E26H 1.33 1.19 0.71 E26 K 1.46 1.47 0.77 E26 L 1.30 1.71 0.77 E26 M 2.001.10 0.89 E26 N 1.37 0.48 0.88 E26 P 0.43 0.99 0.63 E26 R 1.48 0.81 0.77E26 S 1.27 0.28 0.92 E26 T 1.44 0.40 0.82 E26 V 1.39 0.97 0.85 E26 W1.25 0.47 0.68 R27 A 0.45 2.78 0.67 R27 C 0.35 0.58 0.50 R27 E 0.58 0.930.46 R27 G 0.42 0.84 0.24 R27 I 0.72 1.41 0.70 R27 K 1.22 1.55 0.69 R27L 0.48 2.60 0.51 R27 P 0.93 0.48 0.46 R27 R 1.00 1.00 1.00 R27 S 0.530.69 0.56 R27 T 0.41 0.01 0.74 R27 V 0.71 0.94 0.85 R27 W 0.21 −0.590.33 F28 A 1.27 1.48 0.92 F28 C 0.93 1.21 0.87 F28 D 0.67 2.07 0.40 F28E 0.51 1.04 0.85 F28 F 1.00 1.00 1.00 F28 G 0.74 −1.53 0.50 F28 H −0.20−0.19 −0.01 F28 I −0.20 −0.19 −0.01 F28 L 1.09 2.02 0.51 F28 M 1.33 1.370.70 F28 P 0.02 0.39 0.42 F28 R −0.20 −0.19 −0.01 F28 S 1.05 0.70 0.82F28 V 0.86 0.53 0.85 F28 W 1.16 1.17 0.89 F28 Y 0.99 1.36 0.77 A29 A1.00 1.00 1.00 A29 C 1.08 1.15 0.76 A29 D 0.87 1.00 1.06 A29 E 1.12 0.841.02 A29 G 1.60 0.80 1.22 A29 M 0.67 0.77 1.06 A29 P 0.78 0.62 1.07 A29R 1.76 0.73 0.81 A29 S 1.49 0.55 1.05 A29 T 1.42 0.47 1.02 A29 V 1.800.44 1.05 A29 W 1.91 0.74 0.82 A29 Y 1.70 0.59 0.96 P30 A 1.05 0.92 1.15P30 E 1.01 1.24 1.20 P30 G 0.90 1.09 0.99 P30 H 1.01 1.08 1.05 P30 I0.97 1.38 0.95 P30 K 1.21 1.39 1.06 P30 L 0.96 1.17 1.07 P30 M 0.96 0.790.94 P30 P 1.00 1.00 1.00 P30 Q 1.01 0.91 1.06 P30 R 1.16 1.14 0.94 P30S 1.03 1.49 1.12 P30 T 1.05 1.64 1.00 P30 V 1.06 1.74 0.99 P30 Y 0.791.31 1.04 D31 A 1.24 1.18 0.80 D31 D 1.00 1.00 1.00 D31 E 1.13 0.88 0.93D31 F 1.44 1.39 0.65 D31 G 1.44 1.16 0.79 D31 L 1.81 1.61 0.65 D31 N1.34 1.55 0.62 D31 Q 1.07 1.13 0.74 D31 R 1.22 1.49 0.50 D31 S 1.15 1.230.55 D31 T 1.45 1.11 0.76 D31 V 1.28 1.08 0.50 D31 W 1.83 1.14 0.60 V32A 0.43 3.64 1.10 V32 D 0.45 4.19 0.95 V32 E 0.57 3.92 1.00 V32 G 0.582.65 0.98 V32 I 0.91 3.51 1.08 V32 K 1.09 4.73 0.75 V32 L 0.96 4.72 1.01V32 M 0.64 3.41 1.11 V32 N 0.54 1.61 0.99 V32 P 0.01 −1.17 0.31 V32 Q0.64 1.74 1.03 V32 R 1.05 0.72 0.51 V32 S 0.77 1.09 0.85 V32 V 1.00 1.001.00 V32 W 0.94 1.71 0.70 R33 A 0.20 1.32 0.52 R33 C 0.44 1.73 0.95 R33D −0.16 −0.30 −0.02 R33 E −0.16 −0.30 −0.02 R33 G 0.64 2.63 0.47 R33 H−0.16 −0.30 −0.02 R33 K 0.85 2.72 0.81 R33 L 0.34 2.90 0.74 R33 N 0.901.30 0.92 R33 P −0.16 −0.30 −0.02 R33 R 1.00 1.00 1.00 R33 S 1.00 1.010.79 R33 V 0.50 0.94 0.89 R33 W −0.16 −0.30 −0.02 W34 A −0.15 2.29 0.41W34 C −0.15 1.49 0.52 W34 E −0.15 −1.86 0.17 W34 G 0.12 0.88 0.23 W34 I0.18 0.94 0.75 W34 K −0.15 −0.15 −0.02 W34 M 0.16 1.22 0.91 W34 P −0.151.21 0.26 W34 Q 0.02 0.04 0.25 W34 R 0.22 −0.33 0.16 W34 S 0.47 0.080.29 W34 T 0.36 0.15 0.29 W34 V 0.24 0.73 0.71 W34 W 1.00 1.00 1.00 T35A 0.45 3.85 0.98 T35 C 0.55 4.72 1.16 T35 E 0.30 5.73 0.26 T35 I 0.635.38 0.45 T35 K −0.13 −0.54 −0.01 T35 L −0.13 −0.54 −0.01 T35 M 0.172.72 0.40 T35 N 0.20 −2.29 0.43 T35 P −0.13 −0.54 −0.01 T35 Q 0.57 −2.070.52 T35 R 0.18 −11.34 0.23 T35 T 1.00 1.00 1.00 T35 V 0.71 0.34 0.81T35 W −0.13 −0.54 −0.01 T35 Y −0.13 −0.54 −0.01 G36 A 0.63 1.07 1.00 G36C 0.53 1.06 1.09 G36 D −0.12 2.50 0.28 G36 G −0.12 −0.10 −0.02 G36 H0.73 1.10 0.98 G36 I 1.32 1.81 0.31 G36 K 1.27 1.71 0.84 G36 L 1.24 2.490.39 G36 M 0.85 0.54 0.85 G36 N 0.49 0.56 1.08 G36 P −0.12 −0.10 −0.02G36 Q 0.56 0.71 1.07 G36 R 0.99 0.90 0.85 G36 S 0.78 0.26 1.06 G36 T0.76 0.33 0.83 G36 V 0.95 0.38 0.42 G36 W 0.91 0.68 0.57 V37 A 1.25 2.000.63 V37 C 1.09 1.63 0.68 V37 H 1.21 0.96 0.78 V37 I 1.26 1.04 0.77 V37L 1.16 1.16 0.71 V37 N 0.90 1.52 1.09 V37 P 0.53 2.10 0.73 V37 Q −0.11−0.14 −0.02 V37 R −0.11 −0.14 −0.02 V37 S 1.40 1.49 0.81 V37 T 1.05 0.810.63 V37 V −0.11239 −0.14412 −0.02 V37 W 0.92 0.98 0.62 L38 A 0.59 0.630.78 L38 C 0.64 0.72 0.89 L38 D −0.15 0.12 0.24 L38 E −0.15 −0.61 0.26L38 G 0.15 −0.72 0.32 L38 K 0.63 −0.22 0.16 L38 L 1.00 1.00 1.00 L38 P−0.15 −0.78 0.28 L38 Q −0.15 −0.02 0.47 L38 R −0.15 −0.96 0.34 L38 S0.38 0.29 0.48 L38 V 0.88 1.12 0.73 L38 W −0.15 −0.11 −0.02 A39 A 1.001.00 1.00 A39 C 0.63 0.92 0.50 A39 E 1.09 0.83 1.03 A39 F −0.17 −0.11−0.02 A39 G 1.17 0.30 0.92 A39 I 1.26 0.71 0.91 A39 K 1.36 0.96 0.90 A39L 1.43 0.97 0.93 A39 M 0.52 0.81 0.46 A39 N 0.51 0.43 0.45 A39 P 0.690.74 0.45 A39 R 1.17 0.64 0.94 A39 S 0.49 −4.31 0.16 A39 T 1.26 0.790.92 A39 V 1.21 0.98 1.18 A39 W 1.23 1.02 0.94 A39 Y 1.36 1.13 0.90 Q40D 1.16 1.59 0.69 Q40 E 1.08 1.28 0.81 Q40 G 1.79 2.17 0.93 Q40 I 2.581.10 0.49 Q40 K 2.61 3.64 0.52 Q40 L 2.14 1.49 0.53 Q40 N 1.53 1.00 0.78Q40 P 0.45 −0.19 0.24 Q40 Q 1.00 1.00 1.00 Q40 R 1.89 1.48 0.61 Q40 S1.57 1.65 0.87 Q40 T 2.01 1.81 0.75 Q40 W 2.39 2.59 0.54 Q40 Y 1.83 2.020.65 Q41 A 1.03 2.58 0.73 Q41 G 0.97 1.09 0.77 Q41 H 1.12 1.14 0.89 Q41K 1.38 1.61 0.70 Q41 L 1.00 1.92 0.79 Q41 P 0.21 0.66 0.45 Q41 Q 1.001.00 1.00 Q41 R 1.19 1.27 0.74 Q41 S 1.11 0.22 0.92 Q41 V 1.07 −0.050.90 Q41 W 1.14 0.88 0.71 Q41 Y 1.09 0.70 0.82 L42 C 0.76 1.43 0.68 L42D −0.14 −0.17 −0.02 L42 F 1.07 1.02 0.48 L42 G 1.17 0.76 0.50 L42 H 1.92−0.33 0.15 L42 I 0.97 0.66 0.83 L42 K 2.46 1.41 0.13 L42 L 1.00 1.001.00 L42 M 0.78 0.74 0.95 L42 P 0.71 1.34 0.23 L42 Q 0.57 0.28 0.40 L42R 1.38 0.64 0.15 L42 S 0.97 0.45 0.46 L42 T 1.08 −0.04 0.41 L42 V 0.910.73 0.74 L42 W 2.06 −0.70 0.14 G43 A 1.49 1.07 0.45 G43 C 1.48 0.730.36 G43 E 1.25 1.88 0.66 G43 G 1.00 1.00 1.00 G43 H 1.17 0.96 0.63 G43I 0.94 0.77 0.42 G43 K 1.42 0.86 0.65 G43 L 1.22 1.82 0.42 G43 M 1.370.88 0.28 G43 P 1.08 0.31 0.65 G43 Q 0.91 0.48 0.63 G43 R 1.22 0.59 0.57G43 S 1.18 0.23 0.79 G43 V 0.93 0.33 0.44 G43 Y 1.26 0.94 0.36 A44 A1.00 1.00 1.00 A44 C 1.80 1.92 0.46 A44 D −0.17 −0.11 −0.01 A44 E −0.170.03 0.10 A44 F 2.84 0.80 0.99 A44 H −0.17 −0.11 −0.01 A44 L 1.61 0.990.87 A44 M 1.20 0.98 0.71 A44 P −0.17 −0.11 −0.01 A44 R 0.29 −2.17 0.08A44 S 0.52 −0.92 0.16 A44 T 0.30 −1.11 0.14 A44 V 2.13 0.50 0.94 A44 W1.40 0.85 0.61 A44 Y 0.30 −0.23 0.10 D45 A 1.04 0.84 0.99 D45 C 0.830.84 0.48 D45 D 1.00 1.00 1.00 D45 F 1.11 1.04 0.66 D45 G 1.13 0.84 0.94D45 H 1.13 0.78 0.70 D45 K 1.34 0.87 0.86 D45 L 1.05 0.78 0.55 D45 M0.86 0.78 0.88 D45 P 0.75 0.53 0.72 D45 Q 1.04 0.57 0.81 D45 R 1.16 0.490.72 D45 S 1.13 0.38 0.95 D45 T 1.27 0.44 0.86 D45 V 1.05 0.50 0.70 D45W 1.15 0.58 0.54 F46 A 0.92 1.25 1.05 F46 C 0.84 1.16 1.01 F46 D 1.171.39 0.54 F46 E 1.25 1.31 0.38 F46 F 1.00 1.00 1.00 F46 G 1.02 0.94 0.61F46 H −0.13 −0.13 −0.01 F46 I 0.90 0.88 0.91 F46 K 1.00 1.46 0.48 F46 L0.78 1.54 0.74 F46 M 0.78 1.42 0.81 F46 P 0.64 1.50 0.26 F46 S 0.73 0.660.72 F46 T 0.86 0.43 0.79 F46 V 0.82 0.79 0.89 F46 W 0.94 0.63 0.91 E47A 0.95 0.76 0.84 E47 C 0.83 0.77 0.99 E47 D 0.99 0.98 0.97 E47 E 1.001.00 1.00 E47 F 1.09 0.76 0.96 E47 G 1.20 1.10 0.76 E47 H 1.27 0.99 0.93E47 I 1.03 1.15 1.02 E47 K 1.19 1.06 0.89 E47 L 1.00 1.02 0.96 E47 M0.90 0.70 0.84 E47 N 0.91 0.63 0.99 E47 P 1.36 0.36 0.49 E47 R 2.45 0.620.75 E47 S 1.28 0.63 0.83 E47 T 1.96 0.84 0.98 V48 A 0.60 1.63 0.47 V48C 0.83 2.25 0.91 V48 E 0.02 0.99 0.18 V48 F 0.67 1.42 0.57 V48 G 0.610.87 0.25 V48 L 0.92 2.29 0.91 V48 M 0.85 1.79 0.71 V48 N −0.15 0.980.23 V48 P 0.21 3.08 0.34 V48 Q 0.19 1.39 0.32 V48 R 0.76 −1.17 0.15 V48S 0.65 0.42 0.40 V48 V 1.00 1.00 1.00 V48 W −0.15 −0.19 −0.02 I49 A 0.921.87 0.58 I49 E 1.02 0.88 0.75 I49 G 1.34 1.12 0.28 I49 H 1.27 0.74 0.77I49 I 1.00 1.00 1.00 I49 K 1.23 1.26 0.72 I49 L 1.14 1.03 0.93 I49 M1.01 1.02 0.69 I49 P 0.47 0.16 0.29 I49 R 1.05 0.29 0.56 I49 S 1.24 0.790.70 I49 V 1.20 0.97 0.94 I49 W 0.70 0.68 0.64 I49 Y 1.07 1.02 0.82 E50A 1.12 1.23 0.58 E50 D 0.78 1.22 0.80 E50 E 1.00 1.00 1.00 E50 G 0.931.11 0.60 E50 I 0.84 0.58 0.67 E50 L 1.19 0.97 0.41 E50 M 1.18 1.04 0.38E50 P 0.85 1.02 0.71 E50 Q 0.98 0.91 0.70 E50 R 0.46 −0.77 0.20 E50 S0.87 0.65 0.76 E50 V 1.00 0.43 0.81 E50 W 0.75 0.14 0.19 E51 A 1.28 2.720.74 E51 D 0.66 1.28 0.91 E51 E 1.00 1.00 1.00 E51 G 1.22 1.34 0.84 E51I 1.07 0.04 0.52 E51 K 0.38 2.00 0.36 E51 L 1.11 0.93 0.57 E51 M 0.401.20 0.84 E51 P −0.12 −0.39 −0.02 E51 Q 0.98 0.76 0.84 E51 R 0.35 −0.970.29 E51 T 1.18 1.17 0.48 E51 V 1.47 0.37 0.70 E51 W 0.44 0.17 0.22 G52A 0.54 0.79 0.90 G52 E −0.12 0.55 0.41 G52 F −0.12 −0.08 0.52 G52 G 1.001.00 1.00 G52 H 0.18 −0.60 0.49 G52 I 0.10 0.07 0.80 G52 L 0.17 0.240.58 G52 M 0.05 −0.64 0.56 G52 P −0.12 0.24 0.76 G52 Q −0.12 0.28 0.52G52 R −0.12 0.35 0.18 G52 S 0.13 −0.18 0.83 G52 T 0.10 −0.17 0.76 G52 V0.10 −0.16 0.86 G52 W 0.92 2.47 0.13 L53 D 0.01 0.01 0.72 L53 E 0.880.19 0.77 L53 G 1.32 0.33 0.80 L53 H 5.05 1.70 0.27 L53 I 0.55 0.66 0.88L53 K 0.89 0.24 0.70 L53 L 1.00 1.00 1.00 L53 P −0.11 −0.64 0.07 L53 Q1.48 0.72 0.89 L53 R 0.20 −0.02 0.66 L53 S 1.16 0.26 0.95 L53 T 1.020.84 0.75 L53 V 0.52 0.65 0.88 L53 W 0.02 −0.07 0.77 S54 A 3.46 1.411.33 S54 C 1.26 0.88 1.21 S54 D −0.17 0.65 1.08 S54 E −0.17 0.30 1.16S54 F 0.74 −0.14 0.91 S54 G 1.43 0.17 0.93 S54 H −0.17 0.00 1.06 S54 I4.78 0.12 0.94 S54 K 1.44 0.08 0.78 S54 L 2.02 0.26 0.59 S54 M 0.01 0.481.01 S54 N 0.29 1.29 1.01 S54 P 5.20 1.30 0.98 S54 Q 1.03 0.53 0.99 S54R 3.38 0.35 0.84 S54 S 1.00 1.00 1.00 S54 T 1.46 0.33 0.88 S54 V 4.720.29 0.95 S54 W 0.11 −0.07 0.83 S54 Y 0.37 0.12 0.89 A55 A −0.11 −0.15−0.01 A55 C 0.14 1.26 0.98 A55 G 1.69 0.73 0.98 A55 H 0.04 0.92 0.93 A55I 0.34 −0.43 0.80 A55 K 0.52 1.08 0.68 A55 L 0.11 0.87 0.81 A55 N 0.341.05 1.12 A55 P −0.11 −0.01 0.84 A55 R 0.56 0.25 0.99 A55 S 0.76 0.871.08 A55 T 1.69 0.42 0.91 A55 V 0.49 −0.51 0.96 A55 W 0.00 −0.05 0.88A55 Y 0.00 0.18 0.94 R56 A 0.22 0.69 0.85 R56 C 0.45 −0.02 0.93 R56 E−0.12 −0.04 0.16 R56 G 0.30 −0.59 0.56 R56 H −0.12 −0.37 −0.02 R56 K−0.12 −0.37 −0.02 R56 L 0.05 0.24 0.87 R56 N 0.18 0.27 0.31 R56 P −0.12−0.37 −0.02 R56 Q 0.01 −0.01 1.02 R56 R 1.00 1.00 1.00 R56 S 0.39 0.120.55 R56 T 0.10 −0.37 0.85 R56 W −0.12 −0.37 −0.02 R56 Y −0.12 −0.37−0.02 T57 A 0.60 0.65 0.59 T57 C 0.60 0.40 0.85 T57 G 0.92 1.05 0.53 T57H 0.83 0.61 0.23 T57 I 1.19 0.87 0.65 T57 L 0.63 0.76 0.95 T57 N 0.890.25 0.69 T57 P 0.33 −0.87 0.13 T57 R 1.61 −0.66 0.14 T57 S 1.63 1.010.88 T57 T 1.00 1.00 1.00 T57 V 1.28 0.87 0.84 T57 W −0.08 −0.10 −0.01T57 Y 0.52 0.55 0.43 T58 A 0.65 0.36 0.76 T58 E −0.19 −0.10 −0.02 T58 G−0.19 −0.10 −0.02 T58 H 0.89 1.49 0.74 T58 K −0.19 −0.10 −0.02 T58 L0.88 1.12 0.78 T58 M 0.56 0.03 0.50 T58 P −0.19 −0.10 −0.02 T58 R −0.19−0.10 −0.02 T58 S 0.82 0.96 0.90 T58 T 1.00 1.00 1.00 T58 V 0.56 0.961.13 T58 W −0.19 −0.10 −0.02 T58 Y −0.19 −0.10 −0.02 N59 A 0.35 10.440.73 N59 C 0.40 11.23 0.78 N59 D 0.52 11.72 0.67 N59 E 0.66 9.88 0.38N59 F 0.82 10.23 0.57 N59 G 0.88 10.00 0.66 N59 K 0.89 8.21 0.31 N59 L0.88 14.74 0.32 N59 M 0.42 −1.42 0.72 N59 N 1.00 1.00 1.00 N59 P 0.12−55.11 0.14 N59 Q 1.02 1.86 0.73 N59 R 1.09 −11.28 0.39 N59 S 1.06 7.320.74 N59 T 1.07 5.63 0.56 N59 V 0.81 9.97 0.96 N59 W 1.13 12.80 0.59 N59Y 0.80 11.14 0.61 I60 A 0.81 0.79 1.20 I60 C 0.69 0.67 0.97 I60 D 0.830.66 0.56 I60 E 0.87 0.92 0.83 I60 G 1.00 1.04 0.86 I60 H 1.02 1.07 0.96I60 I 1.00 1.00 1.00 I60 K 0.99 0.96 0.73 I60 L 0.95 0.91 1.02 I60 M0.96 0.68 1.14 I60 P 0.23 0.32 0.31 I60 R 1.00 0.81 0.79 I60 S 0.78 1.000.92 I60 V 0.87 1.06 1.06 I60 Y 0.78 1.19 0.89 D61 A 0.70 0.71 1.41 D61C 0.79 0.85 0.92 D61 D 1.00 1.00 1.00 D61 F 1.01 0.70 0.61 D61 G 0.811.25 0.84 D61 H 1.44 1.67 0.97 D61 I 1.08 1.66 0.98 D61 K 0.92 1.72 0.97D61 L 0.80 1.20 1.00 D61 N 0.79 1.00 1.12 D61 P 0.83 1.13 0.97 D61 Q0.89 1.16 1.02 D61 R 1.11 1.59 0.69 D61 S 1.26 1.35 0.97 D61 V 0.95 0.971.10 D61 Y 0.84 0.95 1.03 D62 A −0.24 0.11 1.06 D62 C 0.52 0.49 0.96 D62E 1.02 0.60 0.93 D62 G 0.28 −0.21 0.86 D62 H 0.61 −0.01 0.89 D62 I 0.72−0.25 0.92 D62 L 0.51 −0.37 0.95 D62 M 0.03 −0.24 1.06 D62 P −0.24 −0.550.69 D62 Q −0.24 −0.35 0.86 D62 R 0.12 −0.81 0.62 D62 S 0.57 −0.10 0.88D62 T 0.76 −0.41 0.76 D62 V 0.62 −0.26 0.87 D62 W 0.58 −0.45 0.79 P63 A1.35 0.60 1.06 P63 F 1.25 0.93 0.97 P63 G 1.71 1.22 1.00 P63 K 1.40 1.020.99 P63 L 1.15 1.23 0.84 P63 M 1.46 0.91 1.09 P63 Q 1.09 1.05 1.08 P63R 1.31 0.80 1.02 P63 S 1.42 0.90 1.17 P63 T 1.50 1.32 1.02 P63 V 1.311.04 1.06 P63 W 1.35 1.11 0.86 P63 Y 1.35 0.95 1.12 T64 A 0.96 1.20 0.97T64 C 0.78 0.88 1.05 T64 D 0.87 0.64 0.81 T64 G 1.23 1.08 1.00 T64 H0.89 0.96 0.90 T64 L 0.63 1.22 0.93 T64 M 0.68 1.09 1.07 T64 N 0.69 0.980.91 T64 P 0.76 0.94 0.61 T64 Q 0.76 0.87 1.13 T64 R 0.15 0.11 1.05 T64S 1.11 0.99 1.03 T64 T 1.00 1.00 1.00 T64 W 0.71 0.69 0.72 D65 A 1.310.72 0.72 D65 D 1.00 1.00 1.00 D65 G 0.80 0.52 0.88 D65 H 1.10 0.40 0.71D65 I 0.53 0.62 0.46 D65 P −0.33 0.42 0.08 D65 R 0.41 0.22 0.84 D65 S1.17 0.47 0.76 D65 T 0.90 0.50 0.68 D65 V 0.88 0.20 0.64 D65 W 0.77 0.500.65 D65 Y 0.83 0.42 0.64 P66 A 0.50 0.56 1.03 P66 C 0.51 0.52 1.51 P66D 1.00 0.72 0.90 P66 F 0.95 0.67 1.02 P66 G 1.50 0.44 1.78 P66 H 1.590.95 1.23 P66 I 1.59 0.84 1.51 P66 L 1.14 0.99 0.92 P66 N 1.12 0.38 1.62P66 P −0.09 −0.11 −0.01 P66 Q 1.46 0.42 1.91 P66 R 1.85 0.51 1.26 P66 S1.39 1.02 0.98 P66 T 1.41 1.10 0.72 P66 V 1.83 0.89 1.12 P66 Y 1.33 0.701.08 R67 A −0.20 0.22 1.39 R67 E 1.04 0.11 0.85 R67 F 1.26 0.01 1.01 R67G 1.39 0.41 0.81 R67 K 0.91 0.99 0.76 R67 L 1.20 0.16 1.46 R67 N 1.580.33 1.00 R67 P 1.01 0.04 1.04 R67 Q 1.16 0.13 1.60 R67 R 1.00 1.00 1.00R67 T 1.28 0.32 0.76 R67 V 0.89 0.12 1.24 R67 W 1.07 0.02 0.95 L68 A0.59 −0.11 1.07 L68 C 0.76 0.06 0.85 L68 D −0.16 0.44 0.55 L68 E 1.440.13 0.87 L68 F 0.70 0.25 1.00 L68 G 1.09 −0.08 1.00 L68 H 1.05 0.220.89 L68 I 1.13 0.73 0.86 L68 L 1.00 1.00 1.00 L68 M 0.59 0.03 0.99 L68N 0.51 0.10 0.95 L68 P 0.29 0.35 0.82 L68 Q 0.50 0.25 0.90 L68 R 0.190.47 0.75 L68 S 0.99 0.07 0.93 L68 T 1.03 0.32 0.92 L68 V 1.09 0.51 1.01L68 W 1.21 0.56 0.88 L68 Y 0.71 0.45 0.97 N69 A 0.92 1.13 0.93 N69 C1.05 1.20 1.18 N69 D 0.90 1.11 1.05 N69 G 1.20 0.98 1.06 N69 H 1.36 1.520.73 N69 I 1.47 1.75 0.69 N69 K 1.72 1.59 0.84 N69 L 1.30 1.20 0.36 N69N 1.00 1.00 1.00 N69 P 1.00 0.59 0.66 N69 Q 1.07 1.14 0.74 N69 R 1.490.83 0.84 N69 S 1.21 1.42 1.03 N69 T 1.35 1.43 0.87 N69 V 1.99 1.73 0.87N69 W 1.05 0.55 0.36 N69 Y 0.88 0.17 0.44 G70 A 0.85 1.41 1.08 G70 C0.12 −0.90 0.40 G70 E −0.16 0.33 0.28 G70 F 0.00 −0.36 0.21 G70 G 1.001.00 1.00 G70 H 0.04 1.90 0.26 G70 I 0.04 0.27 0.33 G70 K 0.03 −0.800.26 G70 L 0.03 1.01 0.30 G70 M 0.62 −0.72 0.29 G70 N 0.02 −0.76 0.37G70 P 0.16 −0.58 0.29 G70 Q 0.02 −0.83 0.36 G70 R 0.08 −1.84 0.25 G70 S0.69 0.64 0.88 G70 T 0.27 −0.10 0.45 G70 V 0.16 −0.52 0.34 G70 Y 0.08−0.33 0.38 A71 A 1.00 1.00 1.00 A71 C 1.01 0.99 0.85 A71 D 0.70 0.650.68 A71 E 1.45 0.81 0.83 A71 F 1.13 0.99 0.75 A71 G 1.59 0.68 0.85 A71H 1.70 0.78 0.75 A71 I 1.51 0.79 0.81 A71 K 1.44 1.01 0.76 A71 L 1.230.84 0.85 A71 M 0.98 1.11 0.81 A71 N 1.23 0.61 0.77 A71 P −0.14 −0.050.46 A71 R 1.40 0.77 0.71 A71 S 1.75 0.69 0.84 A71 T 1.70 0.79 0.83 S72A 0.55 3.52 1.06 S72 C 0.56 2.18 0.96 S72 D 0.40 0.80 0.90 S72 E 0.610.93 0.99 S72 F 0.94 1.15 0.80 S72 G 1.20 1.76 0.87 S72 H 1.21 2.48 0.82S72 L 1.26 0.70 1.07 S72 M 0.36 2.13 0.94 S72 N 0.42 2.85 0.99 S72 P−0.25 0.56 0.63 S72 Q 0.62 0.66 0.98 S72 R 0.86 0.74 0.87 S72 S 1.001.00 1.00 S72 T 1.10 0.97 0.88 S72 V 1.08 0.83 0.90 S72 W 0.98 0.34 0.92S72 Y 1.07 0.07 1.03 Y73 A −0.10 1.40 0.82 Y73 C −0.10 1.20 1.18 Y73 D0.13 0.80 1.09 Y73 G 0.71 0.51 0.95 Y73 H 0.67 0.52 0.96 Y73 I 0.82 0.640.97 Y73 K 1.07 0.94 0.95 Y73 L 0.98 0.50 1.03 Y73 M −0.10 1.13 1.05 Y73N 0.56 0.76 1.25 Y73 P 0.64 −0.54 0.42 Y73 Q 1.23 0.87 1.20 Y73 R 1.260.26 0.96 Y73 S 1.17 0.68 0.77 Y73 V 0.88 0.74 1.08 Y73 Y −0.10 −0.10−0.02 L74 A 0.07 2.90 1.01 L74 D −0.18 −0.18 −0.03 L74 F 0.99 1.13 0.58L74 G 1.95 0.57 0.18 L74 H −0.18 −0.18 −0.03 L74 I 0.86 0.64 1.45 L74 L1.00 1.00 1.00 L74 M 0.15 1.21 0.79 L74 P −0.18 −0.18 −0.03 L74 Q −0.18−0.18 −0.03 L74 R −0.18 −0.18 −0.03 L74 S 2.72 −1.52 0.25 L74 T −0.18−0.18 −0.03 L74 V 0.90 0.61 1.18 L74 W 1.38 0.67 0.50 L74 Y 0.90 0.861.19 P75 C 0.54 1.42 1.06 P75 D 0.67 2.09 0.86 P75 E 0.83 1.19 1.00 P75G 1.16 0.93 0.81 P75 H 1.05 0.86 0.89 P75 I 0.69 0.74 0.78 P75 K 0.600.88 0.91 P75 L 0.44 1.19 1.02 P75 M 0.36 0.30 1.22 P75 P 1.00 1.00 1.00P75 Q 1.21 0.61 1.04 P75 R 1.60 0.46 0.89 P75 S 1.39 0.63 1.18 P75 T1.28 0.69 1.10 P75 V 0.93 1.39 0.90 P75 W 1.04 1.31 0.84 P75 Y 0.69 1.321.08 S76 A 0.38 1.11 0.60 S76 C 0.39 1.06 0.67 S76 D 0.41 1.94 0.49 S76E 0.47 2.09 0.58 S76 F 0.44 0.46 0.68 S76 G 0.64 2.15 0.69 S76 H 0.851.11 0.79 S76 K 0.59 1.53 0.32 S76 L 0.74 4.70 0.27 S76 M 0.49 1.61 0.45S76 P 1.23 1.20 0.67 S76 Q 0.84 0.90 0.88 S76 S 1.00 1.00 1.00 S76 T0.75 1.11 0.80 S76 V 0.67 1.35 0.78 S76 W 0.57 −0.25 1.06 S76 Y 0.310.18 0.75 C77 A 0.83 0.91 1.20 C77 C 1.00 1.00 1.00 C77 D 0.92 1.05 0.45C77 F 0.25 −0.61 0.75 C77 G 1.01 0.18 0.53 C77 L 0.98 0.73 1.44 C77 N−0.13 −0.06 −0.04 C77 P −0.13 −0.06 −0.04 C77 R 0.70 −1.02 0.34 C77 S0.95 0.76 1.19 C77 T 1.12 1.03 1.18 C77 V 1.05 0.80 1.33 C77 W 0.39−0.24 0.73 C77 Y 0.95 −0.01 0.66 L78 A −0.11 −0.14 −0.01 L78 C 0.92 0.780.91 L78 E 3.01 −1.14 0.16 L78 G 4.98 1.38 0.12 L78 H 4.82 1.57 0.25 L78I 1.43 1.11 1.06 L78 L 1.00 1.00 1.00 L78 M 0.52 0.48 0.75 L78 N 2.68−0.41 0.22 L78 P −0.11 −0.14 −0.01 L78 Q 1.73 0.52 0.46 L78 R −0.11−0.14 −0.01 L78 S −0.11 −0.14 −0.01 L78 T 1.87 1.10 0.47 L78 V 1.53 0.831.04 L78 Y 1.39 0.81 0.46 A79 A −0.15 −0.13 −0.02 A79 C 0.97 0.03 1.16A79 E 1.12 0.27 1.12 A79 F −0.15 −2.02 0.17 A79 G 0.92 0.92 0.99 A79 H1.93 −0.09 0.85 A79 I 1.59 0.67 0.87 A79 L 1.80 0.96 0.88 A79 M 1.500.28 1.04 A79 N 1.48 0.28 0.97 A79 P 0.70 0.94 0.81 A79 Q 1.47 0.27 1.05A79 R 1.47 0.32 1.02 A79 S 0.82 0.78 1.09 A79 T 1.17 0.60 0.90 A79 V−0.15 −0.13 −0.02 A79 W 1.27 0.53 0.46 T80 A 1.00 1.11 0.90 T80 C 1.311.15 0.91 T80 E 0.07 −0.16 1.02 T80 G 1.16 1.50 0.81 T80 H 0.21 0.050.66 T80 I 0.50 0.15 0.78 T80 K 0.15 −0.32 0.74 T80 L 0.15 −0.11 0.68T80 N 0.53 0.53 0.97 T80 P −0.11 −0.05 0.55 T80 Q 0.91 1.07 1.02 T80 R0.08 −0.22 0.78 T80 S 0.96 1.40 1.12 T80 T 1.00 1.00 1.00 T80 V 1.231.01 0.93 T80 W 0.23 −0.86 0.46 T80 Y 0.15 0.11 0.69 H81 A 1.15 1.450.98 H81 C 1.13 1.09 0.92 H81 F 1.10 0.90 0.87 H81 G 1.17 0.80 0.94 H81H 1.00 1.00 1.00 H81 K 1.52 0.56 0.31 H81 L 1.23 1.03 0.93 H81 M 0.941.54 0.82 H81 N 1.17 1.00 0.82 H81 P −0.10 0.72 0.42 H81 Q 0.85 0.751.00 H81 R 0.34 −0.29 0.85 H81 S 1.04 0.69 0.94 H81 V 1.10 0.71 0.89 H81W 1.13 1.09 0.90 H81 Y 0.77 0.14 0.76 L82 A 0.62 0.98 1.00 L82 G 1.380.31 1.24 L82 H 1.33 0.47 0.95 L82 I 1.17 0.51 0.58 L82 K 1.19 0.51 1.03L82 L 1.00 1.00 1.00 L82 M 0.65 1.06 1.07 L82 P 1.46 0.52 1.11 L82 R1.34 −0.18 1.15 L82 S 1.15 0.00 1.13 L82 T 1.18 0.38 0.97 L82 V 1.020.19 1.14 L82 W 0.27 −0.46 0.93 P83 A 0.36 2.36 0.66 P83 C 0.53 1.010.81 P83 D 0.75 0.83 0.92 P83 E 0.84 1.26 0.92 P83 F 0.76 0.99 0.69 P83G 1.31 0.68 1.01 P83 H 1.27 0.61 0.93 P83 K 1.37 1.16 0.88 P83 L 0.040.21 0.19 P83 M 0.58 1.88 0.71 P83 N 0.70 1.10 0.90 P83 P 1.00 1.00 1.00P83 Q 0.73 0.82 0.95 P83 R 1.19 1.09 0.78 P83 S 1.17 0.79 0.89 P83 T0.86 −0.02 0.62 P83 V 0.78 0.19 0.72 P83 W 0.98 0.62 0.69 L84 A 0.450.45 0.76 L84 D 0.19 0.85 0.48 L84 F 0.72 1.01 0.74 L84 G 0.77 1.01 0.53L84 H 1.01 0.99 0.66 L84 I 0.90 0.87 0.99 L84 K 1.10 0.79 0.59 L84 L1.00 1.00 1.00 L84 N 0.54 0.67 0.86 L84 P −0.12 0.43 0.58 L84 Q 0.410.52 0.93 L84 R 0.56 0.57 0.71 L84 S 0.75 0.55 0.93 L84 T 0.86 0.44 0.95L84 V 0.79 0.42 1.23 L84 W 0.36 −0.28 0.91 D85 A 0.79 1.09 0.63 D85 C0.88 1.50 0.56 D85 D 1.00 1.00 1.00 D85 E 1.12 1.25 0.97 D85 F 1.01 1.980.52 D85 G 1.41 1.60 0.69 D85 H 1.55 1.24 0.76 D85 I 0.55 0.10 0.46 D85L 0.53 0.24 0.52 D85 N 1.54 0.78 0.86 D85 P 0.97 0.54 0.63 D85 Q 3.090.99 0.82 D85 R 2.38 1.03 0.66 D85 S 2.28 0.68 0.93 D85 T 1.33 0.71 0.77D85 V 0.61 0.25 0.65 D85 W 0.87 0.34 0.72 D85 Y 0.98 0.55 0.78 L86 A1.38 3.32 0.40 L86 C 1.16 2.44 0.85 L86 E 0.06 −0.92 0.46 L86 F −0.15−0.26 −0.02 L86 G 1.15 0.70 0.83 L86 H 0.88 −0.72 0.57 L86 L 1.00 1.001.00 L86 P −0.15 0.99 0.22 L86 Q −0.15 −2.60 3.66 L86 R 0.43 −4.46 0.26L86 S 0.78 −0.36 0.78 L86 T 0.96 0.28 0.75 L86 V 0.92 0.12 0.93 L86 W0.67 0.08 0.78 L86 Y 0.85 0.82 0.92 V87 A 0.65 0.17 0.88 V87 C 0.67 2.220.93 V87 D −0.09 −2.53 0.32 V87 F 0.60 0.10 0.56 V87 G 0.46 −2.95 0.54V87 K 0.04 −8.34 0.26 V87 L 0.71 4.30 0.84 V87 M 0.73 0.75 0.86 V87 P0.07 1.64 0.39 V87 R 0.07 −1.33 0.44 V87 S 0.59 −0.09 0.67 V87 T 0.630.15 0.71 V87 V 1.00 1.00 1.00 V87 Y 0.33 −1.24 0.42 I88 G 1.01 −2.630.27 I88 H 1.20 −6.25 0.21 I88 I 1.00 1.00 1.00 I88 M 0.24 1.09 0.86 I88N −0.14 −0.55 0.29 I88 P −0.14 3.51 0.18 I88 Q 0.01 −1.10 0.36 I88 R−0.14 −0.32 −0.02 I88 T 1.03 −0.16 0.52 I88 Y −0.14 −0.32 −0.02 I89 A0.55 1.83 0.63 I89 D −0.10 −0.14 −0.02 I89 E −0.10 −2.05 0.24 I89 F 0.680.75 0.90 I89 G 0.64 −3.84 0.29 I89 H 1.00 −1.01 0.33 I89 I 1.00 1.001.00 I89 L 0.87 1.22 1.07 I89 P 0.38 1.91 0.30 I89 Q 0.25 −0.30 0.32 I89R −0.10 −0.14 −0.02 I89 S 0.71 −1.66 0.49 I89 T 0.94 0.90 0.60 I89 V0.91 0.82 1.09 I89 W 0.53 −2.63 0.27 M90 A 0.78 1.41 0.67 M90 C 0.791.09 0.83 M90 D −0.24 2.88 0.15 M90 E −0.24 1.15 0.29 M90 G 0.57 −1.220.33 M90 I 1.13 0.66 0.74 M90 L 1.02 0.98 0.84 M90 M 1.00 1.00 1.00 M90P −0.24 −0.36 0.28 M90 Q 0.68 0.77 0.71 M90 R −0.24 0.36 0.23 M90 S 1.06−0.17 0.56 M90 T 1.27 0.15 0.59 M90 V 1.08 0.08 0.62 M90 W 0.79 −4.040.21 L91 A 0.57 1.45 0.81 L91 C 0.67 1.27 0.87 L91 D −0.12 1.47 0.12 L91E −0.12 −0.51 0.13 L91 G 1.21 −0.58 0.17 L91 H −0.12 −0.13 −0.01 L91 I0.98 1.05 0.89 L91 K −0.12 −0.13 −0.01 L91 L 1.00 1.00 1.00 L91 M 0.280.88 0.80 L91 P −0.12 −0.13 −0.01 L91 Q 0.05 −0.14 0.18 L91 R −0.12−0.13 −0.01 L91 S 0.92 0.43 0.24 L91 T 1.06 −0.11 0.36 L91 V 0.94 0.790.72 L91 W −0.12 −0.13 −0.01 L91 Y −0.12 −0.13 −0.01 G92 A −0.10 −0.18−0.02 G92 C −0.10 2.05 0.18 G92 D −0.10 −0.18 −0.02 G92 E −0.10 −2.310.21 G92 F −0.10 −3.24 0.17 G92 G 1.00 1.00 1.00 G92 L −0.10 −0.18 −0.02G92 M −0.10 −0.18 −0.02 G92 P −0.10 −0.18 −0.02 G92 R −0.10 −0.18 −0.02G92 S 1.26 −2.96 0.21 G92 T −0.10 −0.18 −0.02 G92 V 1.49 −3.03 0.20 G92W −0.10 −0.18 −0.02 G92 Y −0.10 −0.18 −0.02 T93 A 1.38 1.05 0.50 T93 C1.08 0.95 0.64 T93 D −0.18 0.23 0.22 T93 F 3.52 0.54 0.63 T93 P −0.18−0.19 −0.02 T93 Q −0.18 −6.75 2.03 T93 R −0.18 −0.19 −0.02 T93 S 0.890.49 0.89 T93 T 1.00 1.00 1.00 T93 V −0.18 −0.19 −0.02 T93 W −0.18 −0.19−0.02 T93 Y 5.26 0.03 0.77 N94 A −0.45 0.74 0.96 N94 C 0.01 0.07 0.94N94 G 0.15 0.53 0.76 N94 H 0.11 −0.94 0.77 N94 L 0.61 −0.18 0.49 N94 M−0.45 0.03 0.94 N94 N 1.00 1.00 1.00 N94 P −0.45 0.79 0.40 N94 R 0.10−8.20 0.19 N94 S 0.10 0.88 0.84 N94 T 0.25 −1.43 0.66 N94 V 0.15 −0.390.65 N94 W 0.10 −1.20 0.69 N94 Y 0.08 0.12 0.76 D95 A −0.14 −0.14 −0.01D95 C −0.14 −0.14 −0.01 D95 D 1.00 1.00 1.00 D95 E 2.04 0.75 0.66 D95 G−0.14 −0.14 −0.01 D95 H −0.14 −0.14 −0.01 D95 K −0.14 −0.14 −0.01 D95 L−0.14 −0.14 −0.01 D95 N −0.14 −0.14 −0.01 D95 Q −0.14 −0.14 −0.01 D95 R−0.14 −0.14 −0.01 D95 S −0.14 −0.14 −0.01 D95 T −0.14 −0.14 −0.01 D95 V−0.14 −0.14 −0.01 D95 W −0.14 −0.14 −0.01 D95 Y −0.14 −0.14 −0.01 T96 A0.36 4.20 1.32 T96 C 0.44 3.76 0.79 T96 F 0.53 1.24 0.69 T96 G 0.78 1.281.03 T96 I 0.95 −0.22 0.88 T96 L 0.92 1.93 0.93 T96 M 0.39 2.53 0.80 T96P −0.11 0.89 0.35 T96 R 0.17 0.14 0.50 T96 S 1.04 0.79 1.05 T96 T 1.001.00 1.00 T96 V 0.81 0.59 1.12 T96 W 0.38 −4.29 0.51 T96 Y 0.38 −3.730.59 K97 A 0.01 0.23 1.11 K97 D −0.23 −0.17 −0.01 K97 G 0.84 −0.64 0.39K97 I 0.74 −0.55 0.47 K97 K 1.00 1.00 1.00 K97 L 0.38 −0.28 0.30 K97 M0.02 0.22 0.95 K97 P 0.16 0.27 0.36 K97 Q 1.14 0.00 0.73 K97 R 2.80 0.591.02 K97 S 0.28 −0.46 0.58 K97 T 0.22 −0.42 0.51 K97 V 0.31 −0.45 0.51K97 W 0.42 −2.32 0.13 K97 Y 0.29 −0.65 0.38 A98 A 1.00 1.00 1.00 A98 C1.30 1.42 1.00 A98 D 1.11 2.19 0.81 A98 G 1.57 0.56 0.97 A98 H 2.09 0.920.82 A98 I 2.05 0.65 0.72 A98 L 2.22 1.47 0.71 A98 N 1.24 1.40 1.01 A98P 1.10 1.26 0.90 A98 S 1.73 0.65 1.17 A98 T 1.72 0.27 1.03 A98 Y 2.021.15 0.87 Y99 A 0.66 0.82 1.29 Y99 G 0.83 0.70 1.23 Y99 H 0.77 0.59 1.30Y99 I 0.81 0.61 1.11 Y99 L 0.66 0.86 1.39 Y99 P 0.89 0.81 1.00 Y99 R0.61 0.29 0.97 Y99 S 0.72 0.37 1.45 Y99 V 0.61 0.31 1.28 Y99 W 0.68 0.571.20 Y99 Y 1.00 1.00 1.00 F100 A 0.78 2.02 0.93 F100 C 0.73 1.28 0.78F100 D 0.38 −0.03 0.33 F100 E 1.01 0.15 0.83 F100 F 1.00 1.00 1.00 F100K 0.65 −0.60 0.53 F100 M 0.79 2.19 1.20 F100 N 0.91 1.45 1.12 F100 S0.87 0.85 1.02 F100 T 0.95 1.42 0.71 F100 W 1.08 −0.03 1.06 R101 C 0.710.95 0.96 R101 D 0.85 0.80 1.02 R101 F 0.84 0.97 0.66 R101 I 0.79 0.960.68 R101 K 1.24 0.07 0.90 R101 L 0.83 1.12 1.33 R101 N 0.72 0.92 1.11R101 P 0.50 0.86 0.75 R101 Q 0.86 0.11 1.03 R101 R 1.00 1.00 1.00 R101 V0.74 0.44 0.90 R101 W 0.95 0.00 0.89 R101 Y 0.74 0.80 0.67 R102 A 0.191.79 0.98 R102 C 0.22 0.36 0.78 R102 D 0.01 0.68 0.26 R102 F 0.46 0.230.31 R102 G 0.44 0.27 0.43 R102 L 0.33 1.64 0.95 R102 P −0.07 0.89 0.26R102 Q 0.67 1.19 1.09 R102 R 1.00 1.00 1.00 R102 S 0.46 0.96 0.98 R102 V0.28 0.61 0.80 R102 W 0.29 −1.03 0.34 R102 Y 0.40 1.29 0.70 T103 A 0.97−9.64 0.89 T103 C 0.90 −6.91 0.89 T103 F 0.74 −3.39 0.85 T103 G 1.11−5.27 1.20 T103 H 0.99 −4.15 1.14 T103 I 1.08 −5.15 0.89 T103 K 1.09−4.36 1.05 T103 L 1.05 −1.86 0.88 T103 N 0.77 −6.03 1.07 T103 P 0.69−5.11 1.01 T103 R 0.87 −6.30 0.96 T103 S 0.92 −1.36 1.14 T103 T 1.001.00 1.00 T103 V 0.95 −1.95 0.90 T103 W 1.26 −2.60 0.77 T103 Y 1.19−4.68 0.88 P104 A −0.41 −0.19 −0.04 P104 C 1.95 1.83 1.34 P104 E 1.841.97 1.37 P104 F 1.79 0.86 0.67 P104 G 2.67 0.98 1.25 P104 H 2.84 1.031.11 P104 I 2.43 2.05 1.07 P104 L −0.41 −0.19 −0.04 P104 M 1.09 2.241.01 P104 N 1.62 1.44 1.32 P104 P 1.00 1.00 1.00 P104 Q 1.34 0.85 1.24P104 R 1.62 −0.39 0.83 P104 S 2.48 0.53 1.44 P104 T 2.70 0.33 1.29 P104V 2.59 1.02 1.40 P104 W 2.05 0.23 0.59 L105 A −0.11 −0.18 −0.02 L105 C1.56 1.92 1.05 L105 E −0.11 0.53 0.26 L105 F 1.30 1.73 0.95 L105 G 1.081.40 1.07 L105 H 0.85 1.23 1.07 L105 L 1.00 1.00 1.00 L105 M −0.11 −0.18−0.02 L105 P 1.71 0.90 1.00 L105 Q 0.94 1.04 1.03 L105 R 0.99 1.25 0.94L105 S 0.93 0.61 0.95 L105 T 0.92 0.64 1.00 L105 V 0.15 −0.97 0.37 L105W 1.28 1.71 0.78 L105 Y 0.72 0.62 1.18 D106 A 0.72 1.13 0.69 D106 C 1.011.10 0.80 D106 D 1.00 1.00 1.00 D106 E 1.08 1.09 1.02 D106 F 1.02 1.450.34 D106 G 1.18 1.45 0.67 D106 H 1.09 1.18 0.66 D106 I 1.04 0.92 0.45D106 K 1.28 1.24 0.68 D106 L 1.20 1.00 0.56 D106 M 0.73 0.86 0.77 D106 N0.92 0.64 0.91 D106 P −0.17 0.63 0.18 D106 Q 0.92 0.62 0.94 D106 R 0.980.56 0.91 D106 S 0.98 1.02 0.81 D106 T 1.06 1.38 0.64 D106 V 0.98 1.680.61 D106 W 0.78 1.07 0.34 I107 A 0.81 0.80 0.83 I107 C 0.95 1.41 1.00I107 E 2.55 −0.28 0.21 I107 F 0.99 −0.02 0.19 I107 G 1.76 −10.12 0.25I107 H −0.07 −0.20 −0.02 I107 I 1.00 1.00 1.00 I107 L 0.96 1.04 0.52I107 N 1.81 0.93 0.56 I107 P 0.65 0.32 0.40 I107 Q 0.53 −0.02 0.43 I107R 0.08 −2.75 0.28 I107 S 2.04 1.33 1.05 I107 T 0.64 1.53 0.95 I107 V1.00 0.97 1.04 I107 W −0.07 −0.20 −0.02 I107 Y 0.49 0.52 0.23 A108 A−0.12 −0.07 −0.02 A108 D −0.12 −0.07 −0.02 A108 E 0.14 0.61 0.25 A108 F−0.12 −0.07 −0.02 A108 G 0.99 1.13 1.15 A108 H −0.12 −0.07 −0.02 A108 I−0.12 −0.07 −0.02 A108 K 0.60 2.97 0.31 A108 L 1.41 2.56 0.20 A108 N−0.12 −0.07 −0.02 A108 P −0.12 −0.07 −0.02 A108 Q 0.58 0.73 0.98 A108 R−0.12 −0.07 −0.02 A108 S 0.94 1.00 1.14 A108 T 1.05 0.87 1.08 A108 V0.76 0.95 0.99 L109 A 0.34 0.32 1.07 L109 D 1.00 0.11 1.15 L109 E 0.740.19 1.24 L109 F 0.83 0.32 1.11 L109 G 0.82 0.51 0.88 L109 H 0.85 0.221.06 L109 I 1.05 0.14 1.21 L109 L 1.00 1.00 1.00 L109 M 0.74 0.63 1.00L109 N 1.52 0.66 1.13 L109 P 0.79 0.43 0.35 L109 Q 1.18 0.22 1.08 L109 R0.48 0.21 0.95 L109 S 0.79 0.38 0.94 L109 T 0.63 0.79 0.87 L109 V 0.520.54 1.06 L109 W 1.30 −0.02 0.88 L109 Y 1.16 0.83 0.79 G110 A 0.91 1.010.88 G110 C 0.35 1.43 0.56 G110 D 0.76 1.40 0.87 G110 E 0.26 1.76 0.46G110 F 0.04 2.29 0.30 G110 G 1.00 1.00 1.00 G110 H 0.63 0.73 0.46 G110 I0.06 0.23 0.32 G110 L −0.20 −0.12 −0.02 G110 M 0.16 0.82 0.34 G110 N0.70 0.77 0.89 G110 P 0.02 0.22 0.50 G110 Q 0.44 0.34 0.77 G110 R 0.050.48 0.45 G110 S 0.79 0.30 1.01 G110 T 0.45 −0.05 0.42 G110 W −0.20−1.18 0.20 G110 Y 0.01 −0.88 0.40 M111 A 0.65 1.02 0.89 M111 C 0.92 1.010.95 M111 D −0.27 0.79 0.37 M111 E 0.25 0.67 0.56 M111 F 1.47 0.78 0.75M111 G 0.85 0.32 0.44 M111 H 0.98 0.19 0.40 M111 I 1.95 1.03 0.91 M111 K1.98 0.71 0.58 M111 L 1.55 0.67 0.93 M111 M 1.00 1.00 1.00 M111 N 0.491.31 0.79 M111 P −0.27 0.57 0.39 M111 R 0.27 −0.99 0.34 M111 S 1.03 0.140.52 M111 T 1.49 0.76 0.77 M111 V 1.47 0.93 0.88 M111 W 0.96 1.23 0.30M111 Y 1.43 1.06 0.65 S112 A 0.58 0.94 0.98 S112 E 0.71 1.16 1.05 S112 F0.37 0.88 0.61 S112 H 1.00 0.38 0.93 S112 K 0.84 0.68 0.92 S112 L 1.031.00 0.80 S112 M 0.43 0.56 0.98 S112 N 0.52 0.85 1.09 S112 P −0.19 −0.820.33 S112 R 0.20 −0.44 0.99 S112 S 1.00 1.00 1.00 S112 T 0.95 0.72 0.87S112 V 0.86 0.48 0.73 S112 W 0.74 0.58 0.85 S112 Y 0.68 −0.10 0.90 V113A 0.71 1.31 0.70 V113 C 0.87 0.94 1.06 V113 D 0.78 0.87 0.97 V113 E 0.910.94 0.99 V113 F 1.05 0.96 0.80 V113 G 0.96 0.58 0.89 V113 H 1.34 0.760.84 V113 K 1.19 0.72 0.92 V113 L 1.50 0.85 0.85 V113 M 0.78 1.06 0.93V113 N 0.88 1.22 1.01 V113 P 0.72 1.14 0.65 V113 Q 1.03 1.11 0.94 V113 R1.13 1.11 0.82 V113 S 0.80 0.57 0.91 V113 T 0.94 0.86 0.89 V113 V 1.001.00 1.00 V113 W 0.91 0.80 0.76 V113 Y 1.11 0.98 0.85 L114 A 0.78 1.071.03 L114 C 0.78 1.14 1.10 L114 E 0.32 −0.14 0.42 L114 F −0.11 −0.21−0.02 L114 G 0.96 1.14 0.78 L114 H 0.92 −0.55 0.21 L114 I 0.97 1.17 0.86L114 K −0.11 −0.21 −0.02 L114 L 1.00 1.00 1.00 L114 M 0.73 1.28 1.00L114 N 0.65 0.77 0.95 L114 P 0.30 0.28 0.42 L114 Q 0.59 0.12 0.68 L114 R−0.11 −0.21 −0.02 L114 S 0.87 0.55 0.72 L114 T 0.88 1.05 0.82 L114 V0.91 0.60 0.84 L114 W −0.11 −0.21 −0.02 L114 Y −0.11 −0.21 −0.02 V115 A0.60 1.19 1.11 V115 C 0.73 1.08 1.14 V115 D −0.15 2.21 0.19 V115 F 0.541.69 0.32 V115 G 1.09 1.76 0.43 V115 H −0.15 −0.13 −0.02 V115 I 1.050.99 1.14 V115 K −0.15 −0.13 −0.02 V115 L 1.12 1.30 1.02 V115 M 0.481.32 1.05 V115 P −0.15 2.21 0.26 V115 Q −0.15 1.15 0.32 V115 R 0.10 1.630.21 V115 S 0.95 1.14 0.72 V115 T 1.15 1.28 0.72 V115 V 1.00 1.00 1.00V115 W 1.23 2.48 0.17 V115 Y 1.03 2.07 0.28 T116 A 1.01 0.95 1.08 T116 C0.89 1.05 1.30 T116 E 0.86 0.91 1.29 T116 G 1.10 0.90 1.44 T116 H 1.001.08 1.48 T116 I 0.80 0.76 0.82 T116 L 0.77 0.68 1.03 T116 M 0.83 1.391.28 T116 N 0.93 1.05 1.68 T116 P 0.74 0.84 0.99 T116 Q 0.95 0.77 1.29T116 R 0.64 0.62 1.03 T116 S 0.88 0.96 1.24 T116 T 1.00 1.00 1.00 T116 V0.86 0.57 0.85 T116 W 0.89 0.75 0.96 T116 Y 0.90 0.47 1.09 Q117 A 2.051.73 1.03 Q117 E 1.15 1.21 1.10 Q117 F 1.57 1.02 0.61 Q117 G 2.08 0.790.97 Q117 H 2.33 1.12 1.12 Q117 M 1.54 1.89 0.87 Q117 P −0.25 1.13 0.61Q117 Q 1.00 1.00 1.00 Q117 R 1.56 1.05 1.00 Q117 S 1.95 0.87 1.13 Q117 T2.23 1.10 1.06 Q117 V 2.15 0.76 0.67 Q117 W 2.16 0.71 0.57 Q117 Y 2.231.13 0.76 V118 A 0.84 0.85 1.20 V118 C 0.78 1.14 1.28 V118 D −0.14 0.400.38 V118 E −0.14 −0.43 0.37 V118 F 0.86 1.00 0.89 V118 G 1.08 0.56 0.67V118 I 0.96 0.55 1.01 V118 K 1.13 −2.50 0.28 V118 L 0.93 1.05 0.93 V118M 0.60 0.93 0.90 V118 P 0.12 0.22 0.52 V118 Q 0.38 1.50 0.57 V118 R 0.360.07 0.46 V118 S 0.95 0.82 0.96 V118 T 0.99 0.92 0.90 V118 V 1.00 1.001.00 V118 W 0.83 −1.28 0.42 V118 Y 1.25 1.34 0.60 L119 A 0.81 1.02 1.18L119 C 0.76 0.24 1.18 L119 D 0.24 0.28 0.97 L119 E 0.45 0.32 1.04 L119 F0.56 −0.61 0.93 L119 G 0.93 −0.06 0.97 L119 H 0.91 0.46 0.89 L119 I 0.900.43 1.06 L119 L 1.00 1.00 1.00 L119 N 0.58 0.11 1.14 L119 P −0.14 −0.010.71 L119 R 0.43 −0.66 1.00 L119 S 0.83 −0.17 1.05 L119 T 0.97 0.10 0.94L119 V 0.89 0.15 1.04 L119 W 0.77 0.20 0.88 L119 Y 0.77 0.56 0.89 T120 A0.25 0.66 1.09 T120 C 0.75 0.92 1.14 T120 E 0.58 1.53 1.19 T120 H 0.880.50 1.07 T120 I 0.91 1.56 1.00 T120 K 0.87 1.09 1.12 T120 L 0.80 1.261.00 T120 M 0.05 1.22 0.98 T120 N 0.37 1.42 1.10 T120 P 0.07 −0.45 0.82T120 Q 0.26 0.78 1.05 T120 R 0.24 0.60 0.99 T120 S 1.09 1.07 1.35 T120 T1.00 1.00 1.00 T120 V 0.26 1.07 0.93 T120 Y 0.57 1.61 1.01 S121 A 1.121.55 1.10 S121 C 1.18 1.64 1.09 S121 E 0.89 1.04 1.01 S121 G 1.20 0.991.07 S121 K 1.24 0.78 1.04 S121 L 1.35 1.49 1.12 S121 N 1.14 1.06 1.17S121 P 0.83 0.38 0.92 S121 Q 0.92 1.09 1.01 S121 R 1.26 0.70 1.06 S121 S1.00 1.00 1.00 S121 T 1.13 1.26 0.93 S121 V 1.12 1.59 0.97 S121 W 1.330.77 0.91 A122 A 1.00 1.00 1.00 A122 D 0.26 0.06 0.77 A122 E 0.71 0.471.04 A122 F 0.97 0.15 0.87 A122 G 0.93 −0.42 0.85 A122 H 1.14 0.17 1.00A122 I 1.13 0.65 1.04 A122 K 1.08 0.45 0.96 A122 L 0.93 1.02 1.07 A122 M0.81 0.94 1.06 A122 N 0.83 0.70 1.11 A122 P 0.61 0.55 1.07 A122 Q 0.690.74 1.02 A122 R 0.71 0.40 0.94 A122 S 1.03 0.43 1.05 A122 T 1.08 0.520.97 A122 V 1.04 0.89 1.05 A122 W 0.99 0.86 0.88 G123 A 0.89 1.19 0.96G123 C 0.95 0.30 0.92 G123 D 1.73 0.84 0.90 G123 E 1.13 0.56 0.96 G123 F0.84 0.80 0.85 G123 G 1.00 1.00 1.00 G123 H 1.00 0.74 0.84 G123 K 0.971.12 0.93 G123 L 0.99 1.38 0.79 G123 M 0.84 1.38 0.85 G123 N 0.89 0.710.92 G123 P 1.32 0.81 0.89 G123 Q 0.01 0.31 0.37 G123 R 0.66 0.60 0.83G123 T 1.06 0.54 0.85 G123 V 1.40 0.59 0.89 G123 W 0.95 1.39 0.77 G123 Y0.96 1.24 0.87 G124 A 0.84 0.03 1.20 G124 C 0.72 0.67 1.07 G124 D 0.760.64 0.99 G124 F 1.32 0.95 0.70 G124 G 1.00 1.00 1.00 G124 H 1.59 −0.100.98 G124 I 1.85 −0.08 0.92 G124 L 1.92 0.54 0.98 G124 M 0.97 −0.05 1.36G124 N 0.98 0.60 1.18 G124 P −0.11 −0.08 0.37 G124 Q 1.12 0.21 1.02 G124R 1.14 0.41 0.88 G124 S 1.27 0.56 1.00 G124 T 1.64 0.32 0.97 G124 V 1.440.33 0.93 G124 W 0.73 −0.31 0.84 G124 Y 1.23 0.56 0.66 V125 A 1.69 0.930.91 V125 C 0.96 0.54 0.67 V125 D 1.24 0.54 0.76 V125 E 0.81 0.39 0.73V125 F 0.96 0.63 0.77 V125 G 2.95 1.09 0.60 V125 I 1.01 0.94 1.05 V125 P1.50 0.62 0.83 V125 R 1.30 0.47 0.82 V125 S 1.94 0.79 0.75 V125 V 1.001.00 1.00 V125 W 0.37 0.25 0.48 V125 Y 1.08 0.81 0.82 G126 A 0.96 0.551.02 G126 C 0.35 0.98 0.96 G126 D 0.33 1.22 0.93 G126 E 0.67 0.60 1.02G126 G 1.00 1.00 1.00 G126 I 0.84 0.01 0.81 G126 L 1.17 0.54 0.90 G126 M0.43 1.17 0.92 G126 N 0.38 0.85 1.04 G126 P 1.17 0.67 0.82 G126 R 0.430.76 0.89 G126 S 0.76 0.90 0.90 G126 T 1.58 0.74 0.90 G126 V 0.89 0.180.84 G126 Y 0.54 0.23 0.82 T127 A 0.73 1.10 1.10 T127 C 0.76 0.65 1.04T127 D 0.46 0.62 1.03 T127 E 0.40 −0.01 1.03 T127 G 0.95 0.71 1.04 T127H 1.57 0.60 0.99 T127 I 1.06 0.20 0.91 T127 L 0.90 −0.03 0.94 T127 M0.79 0.64 1.02 T127 P 0.14 0.77 0.95 T127 Q 0.55 0.15 0.86 T127 S 1.050.83 1.08 T127 T 1.00 1.00 1.00 T127 V 1.07 0.68 1.06 T128 A 0.76 1.311.23 T128 D 0.78 0.66 1.14 T128 F 0.79 1.71 1.01 T128 H 0.99 1.08 1.19T128 K 1.06 1.57 1.10 T128 L 1.06 1.72 0.97 T128 M 0.72 1.06 1.28 T128 N0.70 1.36 1.29 T128 P 0.87 1.16 1.18 T128 Q 0.78 1.34 1.24 T128 R 0.871.70 1.03 T128 S 0.92 1.27 1.07 T128 T 1.00 1.00 1.00 T128 V 0.98 1.151.05 T128 W 0.92 1.23 0.95 T128 Y 0.95 1.81 0.96 Y129 A 0.64 0.17 1.39Y129 C 0.66 0.61 1.42 Y129 D 0.35 0.23 1.35 Y129 F 0.71 0.71 1.44 Y129 G0.39 −0.56 1.10 Y129 K 0.31 −0.29 1.00 Y129 L 0.78 0.27 1.22 Y129 M 0.680.21 1.28 Y129 N 0.46 0.53 1.24 Y129 P 0.15 0.59 1.11 Y129 R 0.38 0.181.00 Y129 S 0.67 0.69 1.08 Y129 T 0.46 0.14 1.00 Y129 V 0.24 −0.29 1.00Y129 W 0.47 −0.15 1.01 Y129 Y 1.00 1.00 1.00 P130 A 0.82 0.44 1.03 P130C 0.95 0.64 0.93 P130 E 1.00 0.22 1.08 P130 F 1.08 0.48 0.89 P130 G 1.16−0.19 1.11 P130 H 1.17 0.01 1.00 P130 I 1.12 0.41 0.94 P130 K 1.16 0.551.05 P130 L 1.12 0.09 0.98 P130 M 0.66 0.76 1.03 P130 P 1.00 1.00 1.00P130 R 1.11 0.53 0.95 P130 S 1.16 −0.14 0.96 P130 T 1.19 −0.06 0.96 P130V 1.15 0.37 0.94 P130 W 1.15 0.28 0.80 A131 A 1.00 1.00 1.00 A131 D 1.310.40 0.80 A131 E 1.36 0.97 0.88 A131 G 1.66 0.87 0.83 A131 H 1.72 0.820.75 A131 L 1.83 0.59 0.73 A131 P 1.52 0.71 0.94 A131 Q 1.29 0.74 0.69A131 R 1.76 1.04 0.61 A131 S 1.48 0.68 0.87 A131 V 1.59 0.78 0.89 A131 W1.61 −0.42 0.65 A131 Y 1.50 0.48 0.73 P132 A 0.49 6.08 0.94 P132 C 0.495.68 0.94 P132 D −0.11 −7.16 0.62 P132 E 0.19 3.02 0.80 P132 F 0.76−1.33 0.49 P132 G 0.83 4.98 0.79 P132 H 0.50 −1.95 0.68 P132 I 0.58−3.19 0.64 P132 L 0.87 2.24 0.67 P132 N 0.30 1.05 0.83 P132 P 0.09 6.911.03 P132 Q 0.41 6.15 0.91 P132 R 0.02 −2.19 0.65 P132 S 1.13 5.05 0.96P132 T 0.85 −2.01 0.75 P132 V 0.85 −2.29 0.78 P132 W 0.77 −2.64 0.37P132 Y 1.57 4.78 0.60 K133 A 0.67 0.10 1.01 K133 C 0.56 −0.11 0.72 K133E 0.63 0.76 1.01 K133 F 0.86 0.59 0.73 K133 G 0.97 0.31 0.87 K133 H 1.020.31 0.87 K133 I 0.89 0.45 0.78 K133 K 1.00 1.00 1.00 K133 L 1.05 1.920.76 K133 M 0.68 0.33 0.98 K133 P 0.39 0.71 0.89 K133 Q 0.69 0.52 1.13K133 R 0.78 0.83 1.01 K133 S 0.84 0.58 1.02 K133 T 0.93 0.39 0.97 K133 V0.90 0.23 0.87 K133 W 0.97 0.99 0.46 K133 Y 1.12 1.44 0.75 V134 A 0.751.64 0.87 V134 C 0.77 1.37 0.91 V134 D −0.08 −0.08 −0.02 V134 G 1.711.42 0.45 V134 I 1.12 0.89 0.99 V134 K −0.08 −0.08 −0.02 V134 L 1.131.45 0.78 V134 M 0.82 1.89 0.83 V134 N 1.18 2.80 0.25 V134 P −0.08 1.710.43 V134 Q 0.04 0.79 0.44 V134 R −0.08 −0.08 −0.02 V134 S 1.16 1.440.62 V134 T 1.25 0.86 0.82 V134 V 1.00 1.00 1.00 V134 W −0.08 −0.08−0.02 V134 Y −0.08 −0.08 −0.02 L135 D −0.13 2.90 0.27 L135 E −0.13 0.630.39 L135 F 0.34 −0.03 0.45 L135 G 0.33 −1.71 0.28 L135 K 0.66 −1.230.28 L135 L 1.00 1.00 1.00 L135 M 0.77 0.78 1.01 L135 P −0.13 −1.31 0.22L135 Q 0.34 0.17 0.66 L135 R 0.06 −1.41 0.25 L135 S 0.50 −0.65 0.44 L135T 0.73 −0.42 0.50 L135 V 0.83 0.43 0.82 L135 W 0.71 −0.42 0.36 V136 A0.60 1.60 0.66 V136 C 0.57 1.23 0.87 V136 E −0.09 0.20 0.25 V136 L 0.981.13 1.03 V136 N −0.09 0.40 0.26 V136 P −0.09 −0.12 0.52 V136 R −0.09−0.12 −0.02 V136 T 1.13 1.13 0.68 V136 V 1.00 1.00 1.00 V136 W −0.09−0.12 −0.02 V137 A 1.07 1.46 0.64 V137 C 0.98 1.42 0.85 V137 D −0.17−0.23 −0.01 V137 E −0.17 −0.23 −0.01 V137 F −0.17 −0.23 −0.01 V137 G1.02 0.26 0.13 V137 I 0.98 0.70 0.83 V137 L 1.09 1.27 0.82 V137 M 1.221.13 0.89 V137 N 0.46 −1.29 0.15 V137 P −0.17 −0.23 −0.01 V137 R −0.17−0.23 −0.01 V137 S 0.96 0.29 0.50 V137 T 1.08 0.93 0.73 V137 V 1.00 1.001.00 V137 W −0.17 −0.23 −0.01 V137 Y −0.17 −0.23 −0.01 S138 A 0.69 1.281.44 S138 C 0.64 1.18 1.17 S138 E −0.13 −0.19 −0.02 S138 F −0.13 −0.19−0.02 S138 G 1.05 1.11 1.09 S138 H −0.13 −0.19 −0.02 S138 I 1.15 0.350.56 S138 L −0.13 −0.19 −0.02 S138 M −0.13 −0.19 −0.02 S138 N 0.62 1.310.77 S138 P 0.54 1.39 0.45 S138 Q −0.13 −0.19 −0.02 S138 R −0.13 −0.19−0.02 S138 S 1.00 1.00 1.00 S138 V 1.00 0.69 0.67 S138 W −0.13 −0.19−0.02 S138 Y −0.13 −0.19 −0.02 P139 C 0.08 −0.12 0.18 P139 D −0.13 −1.440.15 P139 E −0.13 −5.11 0.19 P139 F −0.13 −4.13 0.16 P139 G 0.50 −3.080.23 P139 H −0.13 −6.03 0.19 P139 I −0.13 −3.71 0.21 P139 K −0.13 −4.090.12 P139 L −0.13 −0.17 −0.02 P139 N −0.13 −2.11 0.16 P139 P 1.00 1.001.00 P139 Q −0.13 −0.32 0.18 P139 R 0.37 −1.04 0.23 P139 S 0.88 −0.520.43 P139 T 0.01 −3.48 0.15 P139 V −0.13 −1.70 0.17 P139 W −0.13 −0.17−0.02 P139 Y −0.13 −0.17 −0.02 P140 A 1.90 1.83 0.61 P140 C 0.39 1.070.40 P140 D −0.45 −0.23 −0.02 P140 F −0.45 2.89 0.19 P140 G 0.96 3.110.20 P140 H 0.59 2.25 0.23 P140 I 0.45 −1.03 0.24 P140 K −0.45 −0.23−0.02 P140 L −0.45 −0.23 −0.02 P140 M −0.45 −0.23 −0.02 P140 P 1.00 1.001.00 P140 Q −0.45 −1.32 0.32 P140 R −0.45 −2.74 0.25 P140 S 1.31 −1.220.43 P140 T 1.74 −0.78 0.29 P140 V 0.50 −1.12 0.34 P140 W 0.50 −0.970.17 P140 Y 0.32 −1.90 0.24 P141 A 1.10 1.08 1.13 P141 G 1.64 −0.05 1.02P141 H 2.07 0.79 0.93 P141 I 2.29 0.38 0.90 P141 L 2.32 0.65 0.74 P141 N1.32 0.97 0.96 P141 P 1.00 1.00 1.00 P141 Q 1.39 0.37 0.88 P141 R 1.65−0.26 0.61 P141 S 1.70 0.02 0.90 P141 T 1.84 0.12 0.82 P141 V 1.96 0.160.72 L142 A 0.80 0.56 0.67 L142 C 0.74 0.70 0.78 L142 D −0.12 −0.13−0.01 L142 F 1.05 0.54 0.46 L142 G −0.12 −0.13 −0.01 L142 I 0.64 0.281.05 L142 K 1.60 0.66 0.23 L142 L 1.00 1.00 1.00 L142 M −0.12 −0.13−0.01 L142 N −0.12 −0.13 −0.01 L142 P 0.54 0.44 0.48 L142 Q 0.67 0.330.49 L142 R −0.12 −0.13 −0.01 L142 S 0.84 0.31 0.65 L142 T −0.12 −0.13−0.01 L142 V 0.84 0.33 0.82 L142 W 2.41 −1.89 0.16 A143 A 1.00 1.00 1.00A143 C 1.39 1.07 0.81 A143 D 1.45 1.22 0.71 A143 E 1.43 1.13 0.71 A143 F1.56 0.68 0.99 A143 G 1.48 0.42 1.17 A143 H 2.90 1.36 0.70 A143 K 3.161.37 0.62 A143 L 2.51 1.28 0.71 A143 N 1.30 0.82 0.79 A143 P 1.53 0.390.63 A143 Q 1.74 0.81 0.72 A143 R 2.15 0.99 0.62 A143 S 1.77 0.63 0.98A143 T 2.18 0.97 0.74 A143 V 2.45 0.99 0.81 A143 W 2.27 −0.21 0.37 P144A 1.09 0.79 0.91 P144 D 1.45 1.38 0.60 P144 F 1.82 1.08 0.66 P144 G 1.450.62 0.78 P144 H 1.94 1.60 0.66 P144 K 2.09 1.09 0.67 P144 L 1.43 1.150.86 P144 M 1.24 1.01 0.76 P144 N 1.44 1.49 0.74 P144 P 1.00 1.00 1.00P144 Q 1.37 1.08 0.77 P144 R 1.76 1.14 0.68 P144 S 1.69 0.92 0.77 P144 T1.46 0.81 0.80 P144 Y 2.34 1.65 0.70 M145 A 0.44 0.79 0.94 M145 C 1.020.93 0.94 M145 E 0.28 0.48 0.74 M145 F 1.49 0.77 0.95 M145 G 0.48 0.260.92 M145 I 0.79 0.53 1.16 M145 L 1.72 0.61 1.07 M145 M 1.00 1.00 1.00M145 P 0.64 0.78 0.78 M145 Q 0.68 0.57 0.86 M145 R 1.15 0.69 0.78 M145 S0.64 0.78 0.91 M145 T 1.01 0.79 0.91 M145 V 0.72 0.63 1.00 M145 W 1.15−0.13 0.49 M145 Y 0.94 0.82 0.68 P146 A 0.20 1.36 0.73 P146 C 0.31 1.690.62 P146 F 0.55 1.53 0.51 P146 G 0.24 1.04 0.51 P146 H 0.50 1.57 0.56P146 L 0.56 2.00 0.53 P146 M 0.39 1.23 0.79 P146 N 0.37 1.00 0.78 P146 P1.00 1.00 1.00 P146 R 0.36 1.06 0.66 P146 S 0.46 0.96 0.82 P146 T 0.380.76 0.80 P146 V 0.55 0.77 0.89 P146 W 0.56 0.68 0.64 P146 Y 0.35 1.440.54 H147 A 1.28 0.98 0.96 H147 C 0.94 1.17 1.04 H147 D 0.95 1.18 1.00H147 E 1.11 1.10 0.96 H147 G −0.12 −0.15 −0.02 H147 H 1.00 1.00 1.00H147 I 0.89 0.92 0.89 H147 K 0.94 1.06 0.89 H147 L 0.69 1.29 1.09 H147 M0.73 1.44 0.86 H147 N 0.84 1.25 0.98 H147 P 1.12 1.21 0.71 H147 Q 0.711.03 0.86 H147 R 0.89 0.94 0.69 H147 S 1.26 0.75 0.92 H147 T 1.20 0.840.85 H147 V 0.96 0.92 0.90 H147 W 0.88 1.05 0.79 H147 Y 0.75 1.12 0.94P148 A 1.64 1.06 0.96 P148 D 1.03 1.34 0.74 P148 E 1.42 1.19 0.76 P148 F1.37 1.50 0.64 P148 G 0.87 1.20 0.70 P148 K 1.79 1.30 0.72 P148 L 1.641.39 0.74 P148 P 1.00 1.00 1.00 P148 Q 1.33 0.98 0.81 P148 R 1.51 1.250.79 P148 S 1.46 1.21 0.74 P148 T 1.50 1.09 0.79 P148 V 2.43 1.04 0.76P148 Y 1.46 1.37 0.72 W149 A 0.21 0.31 1.35 W149 C 0.18 0.12 0.93 W149 E0.00 −0.04 0.85 W149 F 0.53 0.50 1.27 W149 G 0.26 0.45 1.39 W149 H 0.601.01 0.81 W149 I 0.21 0.24 0.83 W149 L 0.30 0.64 1.06 W149 M 0.33 0.491.32 W149 P −0.32 −0.16 0.92 W149 Q 0.11 0.40 1.10 W149 R 0.04 −0.320.67 W149 S 0.16 0.33 1.28 W149 T 0.26 0.44 0.84 W149 W 1.00 1.00 1.00W149 Y 0.58 0.75 1.15 F150 A 0.01 0.54 1.70 F150 C 0.43 0.78 1.41 F150 E1.23 0.73 1.32 F150 F 1.00 1.00 1.00 F150 G 0.14 0.46 1.13 F150 H 0.531.18 1.09 F150 I 0.40 0.78 1.19 F150 K 0.41 0.85 1.33 F150 L 1.29 1.301.14 F150 M 0.80 0.63 1.69 F150 N 0.55 0.36 1.52 F150 P 0.18 0.32 1.38F150 T 0.37 0.58 1.27 F150 V 0.22 0.51 1.26 F150 W 0.19 0.62 1.26 F150 Y0.72 1.07 1.24 Q151 A 1.29 2.93 0.46 Q151 C 1.05 2.55 0.38 Q151 D 1.472.81 0.83 Q151 E 1.14 2.07 0.99 Q151 F 0.31 −8.08 0.21 Q151 H 1.06 2.190.94 Q151 I 0.08 −2.76 0.16 Q151 K 1.07 2.19 1.04 Q151 L 0.40 −1.53 0.17Q151 M 1.24 6.36 0.24 Q151 P 1.35 1.91 0.50 Q151 Q 1.00 1.00 1.00 Q151 R1.36 2.32 0.68 Q151 S 1.05 2.25 0.86 Q151 T 1.24 2.37 0.64 Q151 V 0.36−1.65 0.25 Q151 W 0.77 0.32 0.33 Q151 Y 1.01 2.75 0.41 L152 A 0.88 1.290.85 L152 C 1.00 1.14 0.87 L152 D 1.07 0.86 0.81 L152 E 1.08 1.23 0.93L152 G 1.08 0.77 0.85 L152 H 1.09 0.92 0.93 L152 I 1.04 0.61 0.77 L152 K1.21 0.91 0.93 L152 L 1.00 1.00 1.00 L152 M 0.99 1.10 0.82 L152 P 0.810.61 0.54 L152 Q 1.07 0.76 0.84 L152 R 1.20 0.91 0.89 L152 S 1.12 0.840.84 L152 T 1.12 0.69 0.82 L152 V 1.22 0.88 0.83 L152 W 1.18 1.55 0.74L152 Y 1.09 1.37 0.89 I153 A 1.19 1.49 0.76 I153 F 1.23 1.75 0.47 I153 H1.46 2.00 0.56 I153 I 1.00 1.00 1.00 I153 K 1.62 2.44 0.43 I153 L 1.271.50 0.82 I153 N 0.72 0.89 1.04 I153 P 0.25 1.87 0.31 I153 S 0.87 1.660.61 I153 T 1.27 1.62 0.64 I153 V 0.96 1.15 0.78 F154 D −0.19 −1.06−0.02 F154 E −0.19 −1.06 −0.02 F154 F 1.00 1.00 1.00 F154 G −0.19 −0.640.17 F154 L −0.19 −1.06 −0.02 F154 P −0.19 −1.06 −0.02 F154 Q 0.39 0.970.45 F154 S 0.13 0.29 0.35 F154 T 0.12 −1.76 0.19 F154 V −0.19 −14.190.18 F154 Y 1.32 4.96 0.92 E155 A 0.99 2.59 0.83 E155 D 1.08 1.24 0.89E155 E 1.00 1.00 1.00 E155 F 1.07 0.23 0.60 E155 G 1.17 1.12 0.82 E155 I0.95 0.65 0.61 E155 K 1.23 1.33 0.83 E155 L 1.31 2.07 0.60 E155 M 0.732.91 0.74 E155 N 0.79 1.79 0.86 E155 P 0.79 2.60 0.65 E155 Q 0.90 0.690.87 E155 R 1.47 −0.07 0.71 E155 S 1.08 1.12 0.82 E155 T 1.49 1.19 0.76E155 V 0.79 0.47 0.63 E155 Y 1.27 2.65 0.55 G156 A 0.99 1.21 0.88 G156 C1.07 1.37 0.84 G156 D 0.96 1.62 0.93 G156 E 0.94 1.14 0.91 G156 F 0.900.73 0.78 G156 G 1.00 1.00 1.00 G156 H 1.04 1.40 0.84 G156 I 0.70 −0.080.44 G156 K 1.10 1.11 0.88 G156 L 0.90 0.94 0.74 G156 M 1.09 1.62 0.80G156 N 1.07 1.38 0.97 G156 P 1.44 1.29 0.59 G156 R 1.05 1.21 0.80 G156 S1.02 1.04 0.88 G156 T 1.15 1.53 0.79 G156 V 0.88 0.97 0.58 G156 W 0.890.90 0.56 G156 Y 0.96 1.40 0.80 G157 A 0.77 0.87 1.00 G157 C 0.96 0.610.92 G157 D 0.93 0.94 0.41 G157 E 0.98 0.84 0.61 G157 F 1.27 1.42 0.61G157 G 1.00 1.00 1.00 G157 H 1.14 1.57 0.70 G157 I 1.11 1.33 0.36 G157 K1.28 1.47 0.46 G157 M 0.96 0.85 0.70 G157 P 0.86 0.01 0.31 G157 R 1.51−0.10 0.42 G157 S 1.30 0.19 0.93 G157 T 1.74 0.99 0.68 G157 V 1.23 0.400.59 E158 A 1.45 1.28 0.91 E158 C 1.46 1.37 0.67 E158 D 1.35 0.89 0.82E158 E 1.00 1.00 1.00 E158 F 2.06 1.77 0.46 E158 H 2.40 1.01 0.59 E158 I1.38 0.94 0.76 E158 K 2.08 1.88 0.62 E158 L 1.59 1.96 0.70 E158 M 1.391.73 0.71 E158 N 1.41 1.58 0.82 E158 P 1.41 1.19 0.85 E158 Q 1.49 1.240.85 E158 R 1.99 1.29 0.62 E158 S 1.57 1.27 0.82 E158 T 1.45 0.91 0.77E158 V 1.52 0.89 0.81 E158 W 1.77 1.31 0.67 E158 Y 1.77 2.48 0.57 Q159 A1.08 0.28 1.13 Q159 C 1.13 0.31 0.79 Q159 D 1.09 0.63 0.90 Q159 E 0.990.97 1.14 Q159 G 0.96 0.72 1.03 Q159 H 0.96 1.48 0.90 Q159 L 1.02 0.700.83 Q159 M 1.07 0.84 0.83 Q159 P 1.06 0.49 0.81 Q159 Q 1.00 1.00 1.00Q159 R 1.15 0.74 0.76 Q159 S 1.10 0.73 0.81 K160 A 0.39 1.14 0.86 K160 C0.48 1.29 0.77 K160 D −0.15 1.19 0.40 K160 G 0.91 0.30 0.56 K160 H 0.980.57 0.65 K160 I 0.97 1.00 0.78 K160 K 1.00 1.00 1.00 K160 L 0.97 0.950.77 K160 M 0.31 1.47 0.78 K160 N 0.37 1.12 0.65 K160 P −0.15 1.66 0.31K160 Q 0.45 1.41 0.75 K160 R 0.83 1.15 0.76 K160 S 0.85 0.70 0.74 K160 W0.89 −0.34 0.21 T161 C 0.84 0.56 1.01 T161 D −0.14 −0.21 −0.02 T161 E−0.14 −0.21 −0.02 T161 G 0.92 0.43 0.94 T161 H 1.82 −0.15 0.42 T161 I1.40 0.98 0.91 T161 L 1.25 1.16 0.81 T161 M 0.57 1.72 0.83 T161 N 0.80−0.86 0.32 T161 P −0.14 −0.21 −0.02 T161 Q 1.04 1.50 0.90 T161 R 3.61−1.68 0.42 T161 S 0.92 0.57 0.98 T161 T 1.00 1.00 1.00 T161 V 1.27 1.241.00 T161 W 1.41 0.00 0.52 T161 Y 2.40 2.62 0.23 T162 C 0.95 3.57 1.17T162 F 0.99 3.23 1.05 T162 G 1.00 1.82 0.88 T162 H 1.02 3.91 1.08 T162 I0.99 2.21 1.16 T162 K 1.22 3.13 0.98 T162 L 1.00 3.59 1.05 T162 M 0.773.49 0.89 T162 N 0.83 3.84 0.98 T162 P 0.96 4.37 0.81 T162 Q 0.93 2.450.89 T162 R 1.17 1.23 0.80 T162 S 0.98 2.01 0.97 T162 T 1.00 1.00 1.00T162 W 1.15 2.04 0.85 T162 Y 1.03 2.89 1.03 E163 A 1.11 1.79 0.73 E163 C1.11 1.08 0.67 E163 D 0.90 1.08 0.82 E163 E 1.00 1.00 1.00 E163 F 1.070.27 0.49 E163 G 1.25 0.80 0.79 E163 H 1.32 0.82 0.69 E163 L 1.50 1.940.58 E163 N 0.91 1.00 0.77 E163 P 0.08 0.77 0.30 E163 R 1.12 0.49 0.72E163 S 1.12 0.85 0.81 E163 V 1.13 0.55 0.69 E163 W 1.21 0.98 0.49 E163 Y1.41 1.89 0.60 L164 A −0.14 −0.85 0.21 L164 C 0.09 0.91 0.63 L164 D−0.14 −0.85 0.12 L164 E −0.14 −0.48 0.18 L164 F 0.50 0.86 0.94 L164 G−0.14 −0.14 0.19 L164 H 0.02 0.12 0.16 L164 L 1.00 1.00 1.00 L164 M 0.691.26 1.09 L164 N −0.14 1.31 0.26 L164 P −0.14 2.41 0.17 L164 Q −0.141.01 0.24 L164 R −0.14 1.61 0.17 L164 S 0.32 1.11 0.25 L164 T 0.82 0.990.52 L164 V 0.87 1.02 1.08 L164 Y 0.43 −1.28 0.20 A165 A 1.00 1.00 1.00A165 C 0.99 1.42 0.97 A165 D 0.89 1.69 0.62 A165 F 1.23 1.00 0.74 A165 G1.05 1.07 1.14 A165 I 1.17 0.59 0.64 A165 K 1.35 0.82 0.78 A165 L 1.081.55 0.70 A165 M 0.97 1.56 0.77 A165 N 1.01 1.20 0.91 A165 P 1.14 1.340.91 A165 Q 1.21 1.32 1.05 A165 R 1.70 1.29 0.87 A165 S 1.00 0.94 1.05A165 T 1.18 1.32 0.83 A165 V 1.21 1.13 0.88 A165 Y 1.20 0.84 0.67 R166 A0.73 1.51 1.12 R166 D 0.56 1.55 1.16 R166 F 1.00 1.10 0.85 R166 G 1.150.91 1.19 R166 H 1.20 1.56 0.97 R166 I 1.26 1.39 0.86 R166 K 1.17 1.201.19 R166 L 1.27 1.50 1.08 R166 M 0.65 1.29 1.26 R166 N 0.75 1.21 1.16R166 P 0.43 1.50 0.97 R166 R 1.00 1.00 1.00 R166 S 1.16 0.95 0.98 R166 T1.19 0.74 1.04 R166 V 1.17 0.76 0.94 R166 W 1.25 1.08 0.80 R166 Y 1.291.22 0.85 V167 A 0.56 4.99 0.98 V167 C 0.79 5.37 1.01 V167 D 0.56 5.540.98 V167 G 0.99 2.83 1.08 V167 H 1.03 2.11 1.12 V167 I 1.08 1.00 1.04V167 L 0.84 2.56 1.13 V167 M 0.53 3.84 1.04 V167 P 0.31 6.08 0.85 V167 Q0.55 2.41 0.97 V167 R 0.78 2.25 0.88 V167 S 0.96 1.86 1.04 V167 T 1.132.47 0.96 V167 V 1.00 1.00 1.00 V167 Y 1.07 2.15 0.94 Y168 C 0.69 −4.730.57 Y168 D −0.11 −1.98 −0.03 Y168 E −0.11 −1.98 −0.03 Y168 F 0.68 5.171.28 Y168 G 1.89 −40.74 0.23 Y168 H −0.11 −1.98 −0.03 Y168 I 0.83 −0.590.90 Y168 K −0.11 −1.98 −0.03 Y168 L 0.59 5.39 1.27 Y168 N −0.11 −1.98−0.03 Y168 P −0.11 −1.98 −0.03 Y168 Q 0.28 −8.27 0.25 Y168 R −0.11 −1.98−0.03 Y168 S −0.11 −1.98 −0.03 Y168 T 1.51 −22.96 0.39 Y168 V 1.19−12.96 0.57 Y168 W −0.11 −1.98 −0.03 Y168 Y 1.00 1.00 1.00 S169 A 0.941.13 0.95 S169 C 1.03 1.38 0.78 S169 I 1.16 1.53 0.66 S169 K 1.21 1.270.94 S169 L 1.08 1.47 0.82 S169 M 0.86 1.40 0.86 S169 P 0.87 0.89 0.69S169 Q 1.02 1.37 0.88 S169 R 1.24 1.19 0.77 S169 S 1.00 1.00 1.00 S169 T1.15 0.97 0.82 S169 Y 1.26 1.10 0.77 A170 A 1.00 1.00 1.00 A170 C 1.151.06 1.02 A170 D 1.27 1.32 0.88 A170 E 1.28 1.17 0.99 A170 F 1.44 1.170.83 A170 G 1.59 0.62 0.96 A170 I 1.59 0.44 0.95 A170 K 1.71 0.83 0.96A170 L 1.05 0.85 0.87 A170 M 1.03 1.28 0.93 A170 N 1.21 1.17 0.96 A170 P0.75 1.33 0.80 A170 Q 1.15 0.89 0.98 A170 S 1.47 0.47 0.99 A170 T 1.400.72 0.86 A170 V 1.20 0.74 0.83 A170 W 1.04 0.83 0.82 A170 Y 0.80 0.890.89 L171 A 0.35 1.66 0.79 L171 C 0.56 1.73 0.97 L171 D −0.06 −0.13−0.01 L171 F 1.30 1.97 0.87 L171 G 1.26 1.33 0.50 L171 H 1.67 1.07 0.61L171 I 1.53 1.42 1.16 L171 K 2.05 1.53 0.31 L171 L 1.00 1.00 1.00 L171 M0.53 2.22 0.90 L171 N 0.96 2.79 0.40 L171 Q 0.97 1.93 0.67 L171 R 0.71−0.20 0.24 L171 S 1.43 1.76 0.72 L171 T 1.54 1.36 0.80 L171 V 1.02 1.390.92 L171 Y 1.20 1.35 0.88 A172 A 1.00 1.00 1.00 A172 C 1.20 0.86 1.09A172 D −0.15 1.42 0.16 A172 E −0.15 −0.44 0.19 A172 G 1.41 0.84 1.07A172 I 1.70 0.58 0.30 A172 K 0.95 −0.43 0.17 A172 L 1.20 1.22 0.70 A172M 0.84 1.06 0.84 A172 N 0.37 0.76 0.30 A172 P −0.15 0.58 0.16 A172 Q0.27 0.18 0.34 A172 R 0.44 −0.18 0.20 A172 S 1.59 0.85 0.96 A172 T 1.250.71 0.85 A172 V 1.40 0.39 0.53 A172 W 1.43 0.45 0.12 A172 Y 0.87 1.760.13 S173 A 0.81 2.72 0.95 S173 C 0.82 3.07 0.59 S173 E 0.78 2.65 0.90S173 F 0.96 2.30 0.71 S173 H 1.07 1.49 0.95 S173 I 0.99 2.22 0.78 S173 K1.17 3.01 0.91 S173 L 1.15 3.86 0.77 S173 M 0.80 3.01 0.84 S173 P 0.192.66 0.35 S173 R 1.09 2.47 0.82 S173 S 1.00 1.00 1.00 S173 T 1.06 1.290.89 S173 V 0.95 2.54 0.75 S173 W 1.16 3.67 0.67 S173 Y 1.19 3.54 0.81F174 A 0.59 2.09 0.61 F174 C 1.32 0.48 0.65 F174 F 1.00 1.00 1.00 F174 G1.60 0.91 0.85 F174 H 0.93 1.05 0.86 F174 K 0.86 1.17 0.76 F174 L 1.051.83 0.82 F174 M 0.91 2.20 0.55 F174 P 1.54 1.46 0.13 F174 Q 1.42 0.460.82 F174 R 0.70 0.52 0.95 F174 S 1.16 0.61 0.75 F174 T 0.80 0.64 0.62F174 V 0.60 0.67 0.82 F174 W 0.96 −0.02 0.85 F174 Y 0.84 1.66 0.77 M175A 0.70 0.66 0.95 M175 E 0.95 1.43 0.89 M175 G 2.04 0.75 0.67 M175 L 1.610.86 1.19 M175 M 1.00 1.00 1.00 M175 N 1.39 1.02 1.11 M175 P −0.20 0.080.16 M175 Q 1.56 0.83 0.98 M175 R 1.55 0.86 1.02 M175 T 2.21 0.90 0.98M175 V 1.93 0.81 1.00 M175 W 1.25 0.76 1.14 M175 Y 0.77 0.72 1.35 K176 A0.42 1.19 0.84 K176 C 0.58 1.01 0.87 K176 D 0.62 1.18 0.74 K176 E 0.671.08 0.88 K176 F 0.36 1.28 0.31 K176 G 1.01 0.73 0.80 K176 K 1.00 1.001.00 K176 L 1.00 0.92 0.58 K176 M 0.56 1.33 0.74 K176 N 0.60 0.94 0.85K176 P 0.01 0.78 0.27 K176 Q 0.59 0.97 1.02 K176 R 0.71 1.03 1.06 K176 S0.76 0.72 0.93 K176 T 1.04 0.97 0.70 K176 V 1.04 1.33 0.71 K176 W 1.191.16 0.41 K176 Y 1.04 0.93 0.60 P178 A 0.31 4.39 0.96 P178 D 0.18 6.440.93 P178 E 0.40 4.15 1.05 P178 G 1.09 2.95 0.67 P178 K 1.34 1.70 0.73P178 L 1.82 7.15 0.53 P178 M 0.53 3.87 0.78 P178 P 0.06 5.02 0.93 P178 Q0.15 3.64 0.93 P178 S 0.62 3.06 0.95 P178 T 0.70 2.28 0.81 P178 V 0.672.70 0.78 P178 W 1.14 0.02 0.64 P178 Y 1.38 6.91 0.74 F179 A −0.18 −0.22−0.02 F179 E 0.02 1.80 0.20 F179 F 1.00 1.00 1.00 F179 G 0.03 1.16 0.36F179 H 0.79 0.93 0.91 F179 L 1.15 1.89 0.43 F179 N 0.77 0.95 0.46 F179 P−0.18 −0.22 −0.02 F179 Q 0.46 −0.87 0.46 F179 R −0.18 −0.22 −0.02 F179 S0.78 0.34 0.62 F179 V 0.70 1.17 0.69 F179 W 0.89 0.86 0.62 F179 Y 1.051.47 0.65 F180 A 0.03 2.70 0.27 F180 C 0.65 1.94 0.66 F180 E −0.14 −0.55−0.02 F180 F 1.00 1.00 1.00 F180 G 0.37 −5.96 0.20 F180 I 1.20 2.11 0.79F180 K 1.08 −6.98 0.24 F180 L 1.30 2.13 0.86 F180 M 0.71 4.36 0.96 F180N −0.14 3.05 0.29 F180 Q 0.21 −1.87 0.36 F180 R 0.64 −3.57 0.26 F180 S0.56 −2.05 0.29 F180 T 1.01 −0.68 0.33 F180 V 1.14 3.24 0.76 F180 W 1.111.81 0.90 F180 Y 1.12 2.99 0.84 D181 A 1.35 1.23 0.65 D181 C 1.09 0.850.56 D181 D 1.00 1.00 1.00 D181 E 1.10 0.72 0.78 D181 F −0.15 −0.17−0.01 D181 G 1.09 0.52 0.37 D181 H −0.15 −0.17 −0.01 D181 I −0.15 −0.17−0.01 D181 K 1.33 0.47 0.41 D181 L 1.25 −0.16 0.16 D181 M −0.15 −0.17−0.01 D181 N −0.15 −0.17 −0.01 D181 P 1.03 0.66 0.60 D181 Q 1.14 0.600.54 D181 R 1.23 0.22 0.45 D181 S 1.21 0.55 0.56 D181 T 1.02 −0.32 0.24D181 V 0.88 −0.34 0.21 D181 W 1.26 −0.52 0.28 D181 Y 1.29 −0.25 0.25A182 A 1.00 1.00 1.00 A182 C 0.97 0.99 1.03 A182 G 0.92 0.94 0.90 A182 H−0.14 −0.18 −0.02 A182 I 0.89 −2.48 0.20 A182 K −0.14 −0.18 −0.02 A182 L−0.14 −0.18 −0.02 A182 M −0.14 −0.18 −0.02 A182 N −0.14 0.53 0.14 A182 P−0.14 −1.13 0.12 A182 Q 0.03 −0.84 0.14 A182 R 0.25 −2.69 0.12 A182 S0.87 0.85 0.90 A182 T 1.14 0.11 0.48 A182 W −0.14 −0.18 −0.02 A182 Y−0.14 −0.18 −0.02 G183 C 0.56 1.99 0.92 G183 D 0.30 0.99 0.62 G183 F0.68 0.19 0.75 G183 G 1.00 1.00 1.00 G183 H 0.98 0.95 0.87 G183 L 0.821.50 0.47 G183 P −0.18 1.02 0.33 G183 Q 0.66 −0.20 0.97 G183 R 0.92 1.090.90 G183 S 0.94 −0.08 1.08 G183 V 0.56 −2.47 0.57 G183 Y 0.97 1.45 0.79S184 A 0.60 1.69 1.31 S184 C 0.81 2.39 1.14 S184 D 0.84 2.24 1.15 S184 E0.94 1.86 1.39 S184 F 1.05 1.27 0.89 S184 G 0.99 0.82 1.15 S184 H 1.020.74 1.07 S184 I 0.92 1.21 0.96 S184 K 0.97 1.61 1.02 S184 L 0.80 2.000.98 S184 M 0.51 1.77 1.25 S184 N 0.64 1.93 1.03 S184 P −0.15 0.85 0.40S184 Q 0.89 1.16 1.09 S184 S 1.00 1.00 1.00 S184 T 1.04 0.60 0.94 S184 V0.80 1.25 1.03 S184 Y 1.06 1.09 0.84 V185 C 0.65 0.83 0.96 V185 D 0.40−2.49 0.21 V185 E 0.73 0.88 0.76 V185 F 1.02 1.20 0.83 V185 G 1.12 −3.670.47 V185 H 1.30 −0.58 0.71 V185 I 1.07 0.63 1.03 V185 K 1.37 0.79 0.66V185 L 1.23 0.93 0.75 V185 M 0.39 1.46 0.77 V185 Q 0.77 1.41 0.73 V185 R1.15 0.79 0.57 V185 S 1.09 0.53 0.75 V185 T 1.11 0.91 0.79 V185 V 1.001.00 1.00 V185 W 1.36 −0.44 0.53 V185 Y 1.37 0.58 0.65 I186 A 1.46 1.790.90 I186 D −0.13 4.29 0.19 I186 F 1.01 0.76 0.77 I186 G 1.86 −5.42 0.35I186 I 1.00 1.00 1.00 I186 K −0.13 −0.36 −0.01 I186 L 1.17 1.14 0.84I186 M 0.86 1.38 1.11 I186 P −0.13 −2.95 0.25 I186 R 0.62 −6.69 0.25I186 S 1.39 −0.21 0.65 I186 T 1.51 0.23 0.79 I186 V 1.28 0.48 0.93 I186W −0.13 −0.36 −0.01 I186 Y −0.13 −0.36 −0.01 S187 A 0.51 1.72 0.86 S187C 0.70 1.67 0.79 S187 D 0.59 1.40 0.82 S187 F 1.02 0.65 0.73 S187 G 1.031.46 0.88 S187 H 1.29 1.51 0.68 S187 I 1.38 1.58 0.78 S187 K 1.45 1.160.76 S187 L 1.37 1.46 0.75 S187 M 0.49 1.87 0.85 S187 N 0.59 1.59 0.90S187 P 0.44 −0.31 0.78 S187 Q 0.63 0.35 0.94 S187 R 1.04 0.55 0.82 S187S 1.00 1.00 1.00 S187 T 1.12 0.23 0.74 S187 V 1.23 0.58 0.89 S187 W 1.300.52 0.73 S187 Y 1.43 0.80 0.76 T188 A 0.97 0.95 1.40 T188 C 0.60 0.872.04 T188 D −0.05 −0.14 −0.02 T188 E 0.24 1.97 0.44 T188 F 0.96 −0.200.63 T188 G 0.93 0.79 1.32 T188 H 1.11 −0.79 0.74 T188 I 1.13 0.10 1.85T188 K −0.05 −0.14 −0.02 T188 L 0.76 0.42 1.76 T188 M 0.49 0.75 1.60T188 N 0.69 1.69 1.24 T188 P −0.05 −0.14 −0.02 T188 Q −0.05 −0.14 −0.02T188 R 1.01 −0.47 1.41 T188 S 1.16 0.91 1.52 T188 T 1.00 1.00 1.00 T188V 1.22 0.15 1.53 T188 W −0.05 −0.14 −0.02 T188 Y 1.48 0.09 0.47 D189 A0.05 1.18 0.53 D189 C 0.19 0.94 0.56 D189 D 0.03 0.89 0.90 D189 E 0.350.77 0.85 D189 F 0.83 0.37 0.63 D189 G 0.80 0.80 0.83 D189 H 1.25 0.950.78 D189 I 0.73 1.27 0.69 D189 L 1.30 1.30 0.61 D189 M 0.06 0.88 0.48D189 N 0.22 0.57 0.80 D189 P −0.12 0.97 0.67 D189 R 0.86 0.39 0.65 D189S 0.88 0.81 0.85 D189 T 1.00 1.21 0.73 D189 V 0.73 0.71 0.72 D189 W 1.090.76 0.60 I194 A 0.29 0.00 1.15 I194 C 0.27 −0.02 1.17 I194 F 0.07 −0.030.95 I194 G 0.10 0.04 0.34 I194 I 1.00 1.00 1.00 I194 L 0.80 0.58 1.32I194 P 0.15 −1.42 0.16 I194 R 0.02 −0.40 0.77 I194 S 0.30 −0.15 0.48I194 V 0.37 0.78 1.03 I194 W 0.04 −0.09 1.12 I194 Y −0.32 −0.01 1.01F196 A −0.13 −0.13 −0.02 F196 C 1.74 1.18 0.70 F196 F 1.00 1.00 1.00F196 G 1.59 −0.30 0.60 F196 H 1.77 −0.24 0.23 F196 I 1.32 1.12 0.81 F196K −0.13 −0.13 −0.02 F196 L 1.77 1.17 1.09 F196 M 1.65 0.71 0.93 F196 N−0.13 −0.13 −0.02 F196 P 0.05 0.39 0.42 F196 Q 1.00 −0.25 0.40 F196 R−0.13 −0.13 −0.02 F196 S 1.58 −1.57 0.29 F196 V 1.40 0.68 0.51 F196 W1.01 0.38 0.88 F196 Y 1.41 0.97 0.73

Example 11 Cloning and Expression of a Sinorhizobium meliloti RSM02162M. smegmatis Perhydrolase Homologue

In this Example, cloning and expression of a S. meliloti perhydrolasehomologue are described. The sequences used in cloning and expressionare provided below. The gene RSM02162 (SEQ ID NO:625) was synthesized byDNA2.0. The gene was given the designation “G00355” and was providedcloned into the commercially available vector, pDRIVE (InvivoGen). Thegene was amplified by PCR from this clone using the primer setG00355rbsF/G00355R, Taq DNA polymerase (Roche) as per the manufacturer'sdirections, with G00355 as the template (10 ng/50 μl reaction) and 10picomoles (per 50 μl reaction) of each primer. The amplification wascarried out in an MJ Research PCR machine using 30 cycles of (1 minuteat 95° C.; 1 minute at 55° C.; and 1 minute at 72° C.). Theamplification of the correct size fragment was confirmed by agarose gelelectrophoresis. The fragment was cloned directly into pCR2.1TOPO(Invitrogen) and transformed into E. coli Top10 cells (Invitrogen).Transformants were selected on L agar containing carbenicillin (100μg/ml) at 37° C. The correct construct was confirmed by sequenceanalysis and designated “pMC355rbs.” FIG. 20 provides a map of thisplasmid.

Primer sequences: G00355rbsF (SEQ ID NO: 626)5′-ggccctaacaggaggaattaaccatggtggaaaaacgttccgttctgtgc-3′ G00355R (SEQ IDNO: 627) 5′-Gcgcgcttagaacagagccgctactttgtcagc-3′ Gene sequence(including stop codon) of RSM02162: (SEQ ID NO: 625) 5′-atggtggaaaaacgttccgttctgtgctttggtgattctctgacttggggctggattccggtgaaagagagctccccaactctgcgttacccatacgaacagcgttggaccggtgctatggctgcacgtctgggtgatggttaccacatcattgaagaaggcctgtccgctcgtactactagcctggacgacccaaacgacgctcgtctgaacggctctacctacctgccgatggctctggcttcacctgccactggatctggtaatcattatgctgggtaccaacgacaccaaaagctactttcatcgtaccccatacgagattgccaacggcatgggtaaactggtaggtcaggtcctgacctgtgcaggtggtgttggtacgccttatccagcaccgaaagtcctggtggttgcacctccaccactggcaccaatgccagatccgtggttcgaaggtatgttcggcggtggttacgagaaatctaaggaactgtccggtctgtacaaagcactggctgatttcatgaaagtggagttcttcgcagcgggtgattgtatctccaccgacggtatcgacggtatccacctgagcgctgaaaccaacatccgcctgggtcatgctattgctgacaaagtagcggctctgttctaa-3′ G00355 Proteinsequence: (SEQ ID NO: 628)MVEKRSVLCFGDSLTWGWIPVKESSPTLRYPYEQRWTGAMAARLGDGYHIIEEGLSARTTSLDDPNDARLNGSTYLPMALASHLPLDLVIIMLGTNDTKSYFHRTPYEIANGMGKLVGQVLTCAGGVGTPYPAPKVLVVAPPPLAPMPDPWFEGMFGGGYEKSKELSGLYKALADFMKVEFFAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF Completesequence of pDRIVEG00355: (SEQ ID NO: 629)gcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagctctaatacgactcactatagggaaagctcggtaccacgcatgctgcagacgcgttacgtatcggatccagaattcgtgattttagaacagagccgctactttgtcagcaatagcatgacccaggcggatgttggtttcagcgctcaggtggataccgtcgataccgtcggtggagatacaatcacccgctgcgaagaactccactttcatgaaatcagccagtgctttgtacagaccggacagttccttagatttctcgtaaccaccgccgaacataccttcgaaccacggatctggcattggtgccagtggtggaggtgcaaccaccaggactttcggtgctggataaggcgtaccaacaccacctgcacaggtcaggacctgacctaccagtttacccatgccgttggcaatctcgtatggggtacgatgaaagtagcttttggtgtcgttggtacccagcataatgattaccagatccagtggcaggtgagaagccagagccatcggcaggtaggtagagccgttcagacgagcgtcgtttgggtcgtccaggctagtagtacgagcggacaggccttcttcaatgatgtggtaaccatcacccagacgtgcagccatagcaccggtccaacgctgttcgtatgggtaacgcagagttggggagctctctttcaccggaatccagccccaagtcagagaatcaccaaagcacagaacggaacgtttttccaccataatctgaattcgtcgacaagcttctcgagcctaggctagctctagaccacacgtgtgggggcccgagctcgcggccgctgtattctatagtgtcacctaaatggccgcacaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactaactcagaatgacttggttgagtactcaccagtcacagaaaagcatcaacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacactgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaacaataaaactgtctgcttacataaacagtaatacaaggggtgttatgagccatattcaacgggaaacgtcttgctctaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgagatgcgctggcagtgttcctgcgccggagcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataaacttttgccattctcaccggattcagtcgtcactcatggtgatttctcacagataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaagaattaattcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagaComplete sequence pMC355rbs: (SEQ ID NO: 630)agcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcttggtaccgagctcggatccactagtaacggccgccagtgtgctggaattcgcccttggccctaacaggaggaattaaccatggtggaaaaacgttccgttctgtgctttggtgattctctgacttggggctggattccggtgaaagagagctccccaactctgcgttacccatacgaacagcgttggaccggtgctatggctgcacgtctgggtgatggttaccacatcattgaagaaggcctgtccgctcgtactactagcctggacgacccaaacgacgctcgtctgaacggctctacctacctgccgatggctctggcactcacctgccactggatctggtaatcattatgctgggtaccaacgacaccaaaagctactttcatcgtaccccatacgagattgccaacggcatgggtaaactggtaggtcaggtcctgacctgtgcaggtggtgttggtacgccttatccagcaccgaaagtcctggtggttgcacctccaccactggcaccaatgccagatccgtggttcgaaggtatgttcggcggtggttacgagaaatctaaggaactgtccggtctgtacaaagcactggctgatttcatgaaagtggagttcttcgcagcgggtgattgtatctccaccgacggtatcgacggtatccacctgagcgctgaaaccaacatccgcctgggtcatgctattgctgacaaagtagcggctctgttctaagcgcgcaagggcgaattctgcagatatccatcacactggcggccgctcgagcatgcatctagagggcccaattcgccctatagtgagtcgtattacaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaattcagggcgcaagggctgctaaaggaagcggaacacgtagaaagccagtccgcagaaacggtgctgaccccggatgaatgtcagctactgggctatctggacaagggaaaacgcaagcgcaaagagaaagcaggtagcttgcagtgggcttacatggcgatagctagactgggcggttttatggacagcaagcgaaccggaattgccagctggggcgccctctggtaaggttgggaagccctgcaaagtaaactggatggctttcttgccgccaaggatctgatggcgcaggggatcaagatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatcccaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagExpression of the Homologue from pMC355rbs

To express the S. meliloti RSM02162 protein from the plasmid pMC355rbs(See, FIG. 20, for a map of this plasmid), a single colony wasinoculated into a 5 mls of L broth containing 100 μg/ml carbenicillinand grown overnight at 37° C. with shaking at 200 rpm. Lysates wereprepared by pelleting the cells from 1 ml of the overnight culture bycentrifugation and lysed with BugBuster (Novagen). The supernatants wereassayed using the pNA activity assay, perhydrolysis assay, and a pNC6assay (to test its ability to hydrolyze carbon chains longer than C4),as described herein.

Assay Results

The following Table (Table 11-1) provides a comparison of the hydrolysisactivity of pNA by G00355 as compared to the M. smegmatis perhydrolase

TABLE 11-1 pNA Hydrolysis Activity pNA Hydrolysis Rate Compared toStrain Rate* Perhydrolase E. coli/pMSATNcoI 85 1 E. coli/pMC355rbs 800.94 E. coli/pCR2.1 34.6 0.41 *Rate is absorbance units/min read at 405nm in a spectrophotometer.

The following Table (Table 11-2) provides a comparison of theperhydrolysis of triacetin by G00355 compared to the M. smegmatisperhydrolase.

TABLE 11-2 Triacetin Perhydrolysis Activity Perhydrolysis ActivityStrain Max Vmax E. coli/pMSATNcoI 1.04 11.88 E. coli/pMC355rbs 1.1725.05 E. coli/pCR2.l 0.1 2.9

The following Table (Table 11-3) provides a comparison of pNC6hydrolysis by G00355 compared to the M. smegmatis perhydrolase.

TABLE 11-3 pNC6 Hydrolysis Activity pNC6 Hydrolysis Rate Compared toStrain Rate* Ms. Perhydrolase E. coli/pMSATNcoI 0.58 1 E. coli/pMC355rbs6.57 11.3 E. coli/pCR2.l 0.47 0.8 *Rate is absorbance units/min read at405 nm in a spectrophotometer.

As these results indicate, the homologue RSM02162 from S. melilotiidentified by amino acid sequence homology to the M. smegmatisperhydrolase demonstrated similar, albeit less perhydrolysis activitythan the M. smegmatis perhydrolase. However, this enzyme exhibiteddifferent substrate specificity, as it was able to hydrolyze pNC6, whilethe wild-type M. smegmatis perhydrolase cannot.

The results of the pNC6 hydrolysis assay indicated that certainpositions/substitutions provided an improvement in the ability of theenzyme to utilize longer chain substrates The positions andsubstitutions identified in preliminary screens are provided in thefollowing Table. It is not intended that the present invention belimited to these specific positions and substitutions, as it iscontemplated that additional positions and/or substitutions will alsoprovide improved activity on longer chain substrates.

TABLE 11-4 Positions/Substitutions with Improved Activity in PNC6 AssayWild-Type Residue/Position Amino Acid Variant(s) L12 G, P, Q S54 L, TI153 F, P F154 Q, S, T, V I194 G F196 A, C, G, I, N, P, Q, S, V

Example 12 Amplification of Genes Encoding M. smegmatis PerhydrolaseHomologues from Environmental Isolates

In this Example, methods used to amplify genes encoding M. smegmatisperhydrolase homologues from environmental isolates are described.

Organisms from soil samples that were positive for thetransesterification reaction were purified to single colonies. Toamplify the genes by PCR, the degenerate primer sets 1AF/5AR and 1eF5iRwere used in a PCR reaction containing isolated chromosomal DNA from 8environmental strains exhibiting the transesterification reaction. ThePCR reaction was carried out using Taq DNA polymerase (Roche) as per themanufacturer's protocol, with 1 μg of chromosomal DNA added as templateand 10 picomoles of each primer in a 50 μl reaction. The reaction wascarried out for 30 cycles of (1 minute at 95° C.; 1 minute at 50° C.,and 1 minute at 72° C.). Since the partial coding sequence of theperhydrolase gene from Mycobacterium parafortuitum was already isolated,the same strain was used as a positive control. The strains weredesignated as: 2G, 2D, 9B, 14B, 18D, 19C, 20A. As indicated below, 20Awas typed as Mycobacterium parafortuitum, and 9B is Mycobacteriumgilvum. Based on protein homology, it was inferred that 2D is also M.parafortuitum and 14B is M. gilvum.

Primer Sequences

1AF: (SEQ ID NO: 631) 5′-gccaagcgaattctgtgtttcggngaytcnyt-3′ 5AR: (SEQID NO: 632) 5′-cgattgttcgcctcgtgtgaartgnrtnccrtc-3′ 1eF: (SEQ ID NO:633) 5′-acggtcctgtgctttggngaytcnyt-3′ 5iR: (SEQ ID NO: 634)5′-ccgctggtcctcatctggrtgntcnccrtc-3′

Amplification with the above primer sets was expected to yield bands ofapproximately 500 bp. In all cases except 2G, the 1AF/5AR primer setproduced a band of the expected size. In the case of 19C, both primersets produced bands of the expected size. The ˜500 bp bands werepurified from agarose gels using a gel purification kit (Qiagen) andanalyzed by sequencing. While the strains 2G and 19C yielded bands ofthe expected size with both primer sets they were not the fragmentsencoding the M. smegmatis perhydrolase homologue.

Partial Sequences of 2D Perhydrolase Homologue and Protein:

Gene: (SEQ ID NO: 635)5′-attctgtgtttcggggattccttgacgtggggatggatccctgtcgaagaaggtgtgcccaccgagcggttcccgcgtgacgtccggtggaccggcgtgctggccgacctgctgggcgaccgctacgaggtgatcgaggaaggcctgtcggcgcgcaccaccaccgccgacgacccggccgacccccggctcaacggttcgcagtatctgccgtcgtgtctggccagccatctgccgctggacctggtgatcctgatgctcggcatcaacgacaccaaggcgaattttggccgcaccccgttcgacatcgccaccggtatgggagtgcttgccacgcaggtgctcaccagcgccggtggcgtggggaccagctatcccgcgccgcaggtgctgatcgtggcgccgccgccgctgggcgagctgccccacccctggttcgacctggtgttctccggcggccgtgagaagaccgccgagttggcccgcgtgtacagcgcgctggcgtcgttcatgaaggtgccgttcttcgacgccggctcggtgatcagcaccgacggcgtggacggcacccacttcacacgaggcgaaacaatcga Protein: (SEQ ID NO:636) ILCFGDSLTWGWIPVEEGVPTERFPRDVRWTGVLADLLGDRYEVIEEGLSARTTTADDPADPRLNGSQYLPSCLASHLPLDLVILMLGINDTKANFGRTPFDIATGMGVLATQVLTSAGGVGTSYPAPQVLIVAPPPLGELPHPWFDLVFSGGREKTAELARVYSALASFMKVPFFDAGSVISTDGVDGTHFTRGETIPartial Sequences of 9B Perhydrolase Homologue and Protein:

Gene: (SEQ ID NO: 637)5′-taccgtcgatgtgtggcctcgtgtgaagtgggtgccgttgccaagcgaattctgtgtttcggggattcgttgacgtggggctggatcccggtcgaggaaggtgtacccacccaacgttttccgaagcgggtgcgctggaccggggtgctggccgacgaactgggtgctggctatgaggttgtcgaggaggggttgagcgcgcgcaccaccaccgctgacgaccctaccgatccccggctgaacggctcggactacctccccgcatgcctggccagccacctgccgctggacctggtgatcctgatgctcgggaccaacgacaccaaggcgaatctgaatcgcacacccgtcgacatcgccagcggaatgggcgtcctggccacccaggtgctcaccagcgcgggcggggtcggcaccagctacccggccccgcaggtgttgatcgtggcaccgccgccgctggccgagatgccgcacccgtggttcgagctggtcttcgacggcggccgggagaagaccgcccaactggcccgggtgtacagcgcgctggcgtcgttcatgaaggtgccgttcttcgacgccggatcggtgatcagcaccgacggtgtcgacggcacccacttcacacgaggcgaaacaatcgac cgg Protein: (SEQ IDNO: 638) GGRCVASCEVGAVAKRILCFGDSLTWGWIPVEEGVPTQRFPKRVRWTGVLADELGAGYEVVEEGLSARTTTADDPTDPRLNGSDYLPACLASHLPLDLVILMLGTNDTKANLNRTPVDIASGMGVLATQVLTSAGGVGTSYPAPQVLIVAPPPLAEMPHPWFELVFDGGREKTAQLARVYSALASFMKVPFFDAGSVIST DGVDGTHFTRGETIDRPartial Sequences of 14B Perhydrolase Homologue and Protein:

Gene: (SEQ ID NO: 639)5′-attctgtgtttcggagattcgttgacgtggggctggatcccggtcgaggaaggtgtacccacccaacgttttccgaagcgggtgcgctggaccggggtgctggccgacgaactgggtgctggctatgaggttgtcgaggaggggttgagcgcgcgcaccaccaccgctgacgaccctaccgatccccggctgaacggctcggactacctccccgcatgcctggccagccacctgccgctggacctggtgatcctgatgctcgggaccaacgacaccaaggcgaatctgaatcgcacacccgtcgacatcgccagcggaatgggcgtcctggccacccaggtgctcaccagcgcgggcggggtcggcaccagctacccggccccgcaggtgttgatcgtggcaccgccgccgctggccgagatgccgcacccgtggttcgagctggtcttcgacggcggccgggagaagaccgcccaactggcccgggtgtacagcgcgctggcgtcgttcatgaaggtgccgttcttcgacgccggatcggtgatcagcaccgacggtgtcgacggcacccacttcacacgagg Protein: (SEQ ID NO: 640)ILCFGDSLTWGWIPVEEGVPTQRFPKRVRWTGVLADELGAGYEVVEEGLSARTTTADDPTDPRLNGSDYLPACLASHLPLDLVILMLGTNDTKANLNRTPVDIASGMGVLATQVLTSAGGVGTSYPAPQVLIVAPPPLAEMPHPWFELVFDGGREKTAQLARVYSALASFMKVPFFDAGSVISTDGVDGTHFTRPartial Sequences of 20A Perhydrolase Homologue and Protein:

Gene: (SEQ ID NO: 641)5′-ttgccaagcggaattctgtgtttcggggattctttgacgtggggatggatccctgtcgaagaaggtgtgcccaccgagcggttcccgcgtgacgtccggtggaccggcgtgctggccgacctgctgggcgaccgctacgaggtgatcgaggaaggcctgtcggcgcgcaccaccaccgccgacgacccggccgacccccggctcaacggttcgcagtatctgccgtcgtgtctggccagccatctgccgctggacctggtgatcctgatgctcggcatcaacgacaccaaggcgaattttggccgcaccccgttcgacatcgccaccggtatgggagtgcttgccacgcaggtgctcaccagcgccggtggcgtggggaccagctatcccgcgccgcaggtgctgatcgtggcgccgccgccgctgggcgagctgccccacccctggttcgacctggtgttctccggcggccgtgagaagaccgccgagttggcccgcgtgtacagcgcgctggcgtcgttcatgaaggtgccgttcttcgacgccggctcggtgatcagcaccgacggcgtggacggcacccacttcacacgaggc gaaacaatcga-3′Protein: (SEQ ID NO: 642)LPSGILCFGDSLTWGWIPVEEGVPTERFPRDVRWTGVLADLLGDRYEVIEEGLSARTTTADDPADPRLNGSQYLPSCLASHLPLDLVILMLGINDTKANFGRTPFDIATGMGVLATQVLTSAGGVGTSYPAPQVLIVAPPPLGELPHPWFDLVFSGGREKTAELARVYSALASFMKVPFFDAGSVISTDGVDGTHFTRG ETIIdentification of the Natural Isolates

To type the environmental isolates used in this Example, plates of thepurified strains were sent to MIDI for 16S rRNA typing. 20A isMycobacterium parafortuitum, 9B is Mycobacterium gilvum. By proteinhomology we infer that 2D is also M. parafortuitum and 14B is M. gilvum.

Example 13 Sequence and Taxonomic Analyses of Perhydrolase Homologues

In this Example, sequence and taxonomic analyses of M. smegmatisperhydrolase homologues are provided

Taxonomic Assignment

The basic “List of 60” protein sequences accessed from public databasesand used for construction of primer sets for screening of metagenomiclibraries (BRAIN) was converted into a document illustrating themicrobial taxonomic origins of the proteins, as described below. Thisinformation was used to produce the following alignment.

1                                               50 MSAT (1)-------------MAKRILCFGDSLUWGWVPVEDGAPU-ERFAPDVRWUG 14B natural isolate(1) -----------------ILCFGDSLTWGWIPVEEGVPT-QRFPKRVRWTG 20A (1)-------------LPSGILCFGDSLTWGWIPVEEGVPT-ERFPRDVRWTG 2D natural isolate(1) -----------------ILCFGDSLTWGWIPVEEGVPT-ERFPRDVRWTG 9B NaturalIsolate (1) -GGRCVASCEVGAVAKRILCFGDSLTWGWIPVEEGVPT-QRFPKRVRWTG M.parafortuitum CO1 (1) -------------MAKRILCFGDSLTWGWIPVEEGVPT-ERFPRDVRWTGSm-RSM05666 (1) --------------MKTVLCYGDSLTWGYDATGSG-----RHALEDRWPSAt-Q8UAC0 (1) --------------MKTVLAFGDSLTWGADPATG---L--RHPVEHRWPDAt-Q8UFG4 (1) -------------MVKSVLCFGDSLTWGSNAETGG-----RHSHDDLWPSM091_M4aE11 (1) --------------MKTILAYGDSLTYGANPIPGGP----RHAYEDRWPTM1-RML000301 (1) MAGGTRLDECTGERMKTVLCYGDSLTWGYNAEGG------RHALEDRWPSP.dejongeii RVM04532 (1)--------------MKTILCFGDSNTWGYDPASMTAPFPRRHGPEVRWTG Q92XZ1 Sinorhizobiummeliloti (1) ---------MEETVARTVLCFGDSNTHGQVPGRGPLDR---YRREQRWGG Q98MY5Mesorhizobium loti (1)--------------MKTVLCYGDSLTWGYNAEGG------RHALEDRWPS RSM02162_Sm (1)-----------MVEKRSVLCFGDSLTWGWIPVKESSPT-LRYPYEQRWTG S261_M2aA12 (1)--------------MKNILAFGDSLTWGFVAGQDAR-----HPFETRWPN Sma1993 Sinorhizobium(1) MTINSHSWRTLMVEKRSVLCFGDSLTWGWIPVKESSPT-LRYPPEQRWTG melilotiConsensus (1)                KTILCFGDSLTWGWIPV EG P   RHP E RW G51                                             100 MSAT (37)VLAQQLGADFEVIE--EGLSARUUNIDDPUDPRL-NGASYLPSCLAUHLP 14B natural isolate(33) VLADELGAGYEVVE--EGLSARTTTADDPTDPRL-NGSDYLPACLASHLP 20A (37)VLADLLGDRYEVIE--EGLSARTTTADDPADPRL-NGSQYLPSCLASHLP 2D natural isolate(33) VLADLLGDRYEVIE--EGLSARTTTADDPADPRL-NGSQYLPSCLASHLP 9B NaturalIsolate (49) VLADELGAGYEVVE--EGLSARTTTADDPTDPRL-NGSDYLPACLASHLP M.parafortuitum CO1 (37)VLADLLGDRYEVIE--EGLSARTTTAEDPADPRL-NGSQYLPSCLASHLP Sm-RSM05666 (32)VLQKALGSDAHVIA--EGLNGRTTAYDDHLADCDRNGARVLPTVLHTHAP At-Q8UAC0 (32)VLEAELAGKAKVHP--EGLGGRTTCYDDHAGPACANGARALEVALSCHMP At-Q8UFG4 (33)VLQKALGSDVHVIFTHEGLGGRTTAYDDHTGDCDRNGARLLPTLLHSHAP M091_M4aE11 (33)ALEQGLGGKARVIA--EGLGGRTTVHDDWFANADANGARVLPTLLESHSP M1-RML000301 (45)VLQASLGGGVQVIA--DGLNGRTTAFDDHLAGADANGARLLPTALTTHAP P.dejongeii RVM04532(37) VLAKALGAGFRVIE--EGQNGRTTVHEDPLNICR-KGKDYLPACLESHKP Q92XZ1Sinorhizobium meliloti (39)VLQGLLGPNWQVIE--EGLSGRTTVHDDPIEGSLKNGRIYLRPCLQSHAP Q98MY5 Mesorhizobiumloti (31) VLQASLGGGVQVIA--DGLNGRTTAFDDHLAGADANGARLLPTALTTHAP RSM02162_Sm(39) AMAARLGDGYHIIE--EGLSARTTSLDDPNDARL-NGSTYLPMALASHLP S261_M2aA12 (32)ALAAGLGGKARVIE--EGQNGRTTVFDDAATFESRNGSVALPLLLISHQP Sma1993 Sinorhizobium(50) AMAARLGDGYHIIE--EGLSARTTSLDDPNDARL-NGSTYLPMALASHLP melilotiConsensus (51) VLA  LGG Y VIE  EGLSGRTT  DDP D  L NGS YLPT  LASHLP101                                            150 MSAT (84)LDLVIIMLGUNDUKAYFRRUPLDIA--LGMSVLVUQVLUSAGGVGUUYPA 14B natural isolate(80) LDLVILMLGTNDTKANLNRTPVDIA--SGMGVLATQVLTSAGGVGTSYPA 20A (84)LDLVILMLGINDTKANFGRTPFDIA--TGMGVLATQVLTSAGGVGTSYPA 2D natural isolate(80) LDLVILMLGINDTKANFGRTPFDIA--TGMGVLATQVLTSAGGVGTSYPA 9B NaturalIsolate (96) LDLVILMLGTNDTKANLNATPVDIA--SGMGVLATQVLTSAGGVGTSYPA M.parafortuitum CO1 (84)LDLVILMLGTNDTKANFGRTPFDIA--TGMGVLATQVLTSAGGVGTSYPA Sm-RSM05666 (80)LDLIVFMLGSNDMKPIIHGTAFGAV--KGIERLVNLVRAHDWPTETE-EG At-Q8UAC0 (80)LDLVIIMLGINDIKPVHGGRAEAAV--SGMARLAQIVETFIYKPREA--V At-Q8UFG4 (83)LDMVIIMLGTNDMKPAIHGSAIVAFTMKGVERLVKLTRNHVWQVSDW-EA M091_M4aE11 (81)LDLIVIMLGTNDIKPHHGRTAGEAG--RGMARLVQIIRGHYAGRMQD--E M1-RML000301 (93)IDLIVIMLGANDMKPWIHGNPVAAK--QGIQRLIDIVRGHDYPFDWP--A P.dejongeii RVM04532(84) LDLVILMLGTNDLKSTFNVPPGEIA--AGAGVLGRMILAGDAGPENR--P Q92XZ1Sinorhizobium meliloti (87)LDLIIIMLGTNDLKARFNMPPSEVA--MGIGCLVHDIRELSPGRTGN--D Q98MY5 Mesorhizobiumloti (79) IDLIVIMLGANDMKPWIHGNPVAAK--QGIQRLIDIVRGHDYPFDWP--A RSM02162_Sm(86) LDLVIIMLGTNDTKSYFHRTPYEIA--NGMGKLVGQVLTCAGGVGLPYPA S261_M2aA12 (80)LDLVIIMLGTNDIKFAARCRAFDAS--MGMERLIQIVRSANYMKGYK--I Sma1993 Sinorhizobium(97) LDLVIIMLGTNDTKSYFHRTPYEIA--NGMGKLVGQVLTCAGGVGTPYPA melilotiConsensus (101) LDLVIIMLGTNDMKA   RTP DIA   GMGRLV  VLT AGGVG    A151                                            200 MSAT (132)PKVLVVSPPPLAPM-PHPWFQLIF-EGGEQKUUELARVYSALASFMKVPF 14B natural isolate(128) PQVLIVAPPPLAEM-PHPWFELVF-DGGREKTAQLARVYSALASFMKVPF 20A (132)PQVLIVAPPPLGEL-PHPWFDLVF-SGGREKTAELARVYSALASFMKVPF 2D natural isolate(128) PQVLIVAPPPLGEL-PHPWFDLVF-SGGREKTAELARVYSALASFMKVPF 9B NaturalIsolate (144) PQVLIVAPPPLAEM-PHPWFELVF-DGGREKTAQLARVYSALASFMKVPF M.parafortuitum CO1 (132)PQVLIVAPPPLGEL-PHPWFDLVF-SGGREKTAELARVYSALASFMKVPF Sm-RSM05666 (127)PEILIVSPPPLCET--ANSAFAAMFAGGVEQSAMLAPLYRDLADELDCGF At-Q8UAC0 (126)PKLLIVAPPPCVAG---PGGEPAG-GRDIEQSMRLAPLYRKLAAELGHHF At-Q8UFG4 (132)PDVLIVAPPQLCETANPFMGAIFRDAIDESAMLASVFTYRDLADELDCGF M091_M4aE11 (127)PQIILVSPPPIILGDWADMMDHFGPHEAIATSVDFAREYKKRADEQKVHF Ml-RMLO00301 (139)PQILIVSPPVVSRT--ENADFREMFAGGDEASKQLAPQYAALADEVGCGF P. dejongeii RVM04532(130) PQLLLMCPPKVRDLSAMPDLDAKI-PHGAARSAEFPRHYKAQAVALKCEY Q92XZ1Sinorhizobium meliloti (133)PEIMIVAPPPMLED--LKEWESIF-SGAQEKSRKLALEFEIMADSLEAHF Q98MY5 Mesorhizobiumloti (125) PQILIVSPPVVSRT--ENADFREMFAGGDEASKQLAPQYAALADEVGCGFRSM02162_Sm (134) PKVLVVAPPPLAPM-PDPWFEGMF-GGGYEKSKELSGLYKALADFMKVEFS261_M2aA12 (126) PEILIISPPSLVPT--QDEWFNDLWGHAIAESKLFAKHYKRVAEELKVHFSma1993 Sinorhizobium (145)PKVLVVAPPPLAPM-PDPWFEGMF-GGGYEKSKELSGLYKALADFMKVEF meliloti Consensus(151) PQVLIVAPPPL EM   P FE VF  GG EKS  LARVY ALAD MKV F201                                   241 MSAT (180)FDAGSVISUDGVDGIHFUEANNRDLGVALAEQVRSLL---- (SEQ ID NO: 643) 14B naturalisolate (176) FDAGSVISTDGVDGTHFIR---------------------- (SEQ ID NO: 644)20A (180) FDAGSVISTDGVDGTHFTRGETI------------------ (SEQ ID NO: 645) 2Dnatural isolate (176) FDAGSVISTDGVDGTHFTRGETI------------------ (SEQ IDNO: 646) 9B Natural Isolate (192)FDAGSVISTDGVDGTHFTRGETIDR---------------- (SEQ ID NO: 647) M.parafortuitum CO1 (180) FDAGSVISTDGVDGIHFTRGEQST----------------- (SEQID NO: 648) Sm-RSM05666 (175) FDGGSVARTTPIDGVHLDAENTRAVGRGLEPVVRMMLGL--(SEQ ID NO: 649) At-Q8UAC0 (172)FDAGSVASASPVDGVHLDASATAAIGRALAAPVRDILG--- (SEQ ID NO: 650) At-Q8UFG4(182) FDAGSVARTTPVDGVHLDAENTRAIGRGLEPVVRMMLGL-- (SEQ ID NO: 651)M091_M4aE11 (177) FDAGTVATTSKADGIHLDPANTRAIGAGLVPLVKQVLGL-- (SEQ ID NO:652) Ml-RML000301 (187) FDAGTVAQTTPLDGVHLDAENTRNIGKALTSVVRVML---- (SEQID NO: 653) P. dejongeii RVM04532 (179)FNSQEIVETSPVDGIHLEASEHLKLGEALAEKVKVLLG--- (SEQ ID NO: 654) Q92XZ1Sinorhizobium meliloti (180) FDAGTVCQCSPADGFHIDEDAHRLLGEALAQEVLAIGWPDA(SEQ ID NO: 655) Q98MY5 Mesorhizobium loti (173)FDAGTVAQTTPLDGVHLDAENTRNIGKALTSVVRVMLEL-- (SEQ ID NO: 656) RSM02162_Sm(182) FAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF---- (SEQ ID NO: 657)S261_M2aA12 (174) FDAGTVAVADKTDGGHLDAVNTKAIGVALVPVVKSILAL-- (SEQ ID NO:658) Sma1993 Sinorhizobium (193)FAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF---- (SEQ ID NO: 659) melilotiConsensus (201) FDAGSVISTD VDGIHLDA  T  IG AL   VR LL (SEQ ID NO: 660)

The alignment tree from the CLUSTALW alignment (which approximates to aphylogenetic tree) suggests 3 or 4 groupings.

From this alignment, a hypothetical protein sequence was constructedfrom the consensus sequence. Where no consensus existed the site wasfilled with the Per amino acid; gaps were ignored. This provided aPer-consensus sequence:

(SEQ ID NO: 661) 1 TILCFGDSLT WGWIPVEEGA PTERHPPEVR WTGVLAQQLGGDYEVIEEGL 51 SGRTTNIDDP TDPRLNGSSY LPTCLASHLP LDLVIIMLGT NDMKAYFRRT 101PLDIALGMGR LVTQVLTSAG GVGTTYPAPQ VLIVAPPPLA EMPHPWFELV 151 FEGGEEKSTELARVYSALAD FMKVPFFDAG SVISTDGVDG IHLDAANTRD 201 IGVALAEQVR SLL

This consensus sequence was used for a BLASTP search against anon-redundant database. This search identified 55 hits. The majority ofthe ‘hits’ were GDSL or GDSI type molecules covering a wide range ofmicrobial diversity. However, only the first 14 ‘hits’ had e-values andbit-values in the reliable range. At first sight, this appeared toprovide further molecules with a GDSL/N-G/ARTT motif, but this was foundto be due to differences in coding (Swiss Prot vs GenBank)

The screening of 3 environmental libraries (at BRAIN) resulted in 10clones with a GDSL motif. A further 2 clones were derived from the BRAINlibrary. The following Table (Table 13-1) lists the clones and indicatestheir activity.

TABLE 13-1 Clones with GDSL Motifs Library Clone Perhydrolase ActivityS248Fa S248_M40cD4 No S248Fa S248_M44aA5 No S248Fa S248_M18bH12 NotPerhydrolase S248Fa S248_M36bC5 Not Perhydrolase S248Fa S248_M50cD9 NotPerhydrolase S248Fa S248_M2bB11 ? Low S261 S261_M2aA12 Yes S279S279_M75bA2 Not done S279 S279_M11aC12 Not GDSL S279 S279_M70aE8 ? LowM091 M091_M4aE11 Not tested BRAIN Est114 No BRAIN Est105 Not doneM40cD4

Strongest hit: arylesterase of Brucella melitensis (46% identical).Motifs: GDSL-GAND; GQTT instead of GRTT. Sequence alignment against thecore list of organisms places it close to Caulobacter vibrioides andBrucella melitensis in the alpha-Proteobacteria.

M44aA5

Strongest hit: Acyl-CoA thioesterase of Pseudomonas aeruginosa (43%identical). Motifs: GDSL-GGND; no GRTT or equivalent. Sequence alignmentagainst the core list of organisms places it close to Pseudomonas sp inthe gamma-Proteobacteria.

M2bB11

Strongest hit: arylesterase of Brucella melitensis. Motifs: GDSL-GAND;no GRTT or equivalent. Sequence alignment against the core list oforganisms shows no strong association placing it between the alpha- andgamma-Proteobacteria.

M2aA12

Strongest hit: arylesterase of Agrobacterium tumefaciens (42% identical)Motifs: GDSL-GRTT-GTND. Sequence alignment against the core list oforganisms places it close to Agrobacterium tumefaciens in thealpha-Proteobacteria.

M75bA2

Strongest hit: incomplete. BLAST search revealed nothing significant.Motifs: GDSL-GTND; no GRTT or equivalent. Sequence alignment against thecore list of organisms shows no convincing associations. The closestneighbors appear to be the Vibrio-Aeromonas groups of thegamma-Proteobacteria.

M70aE8

Strongest hit: acyl-CoA thioesterase from E. coli (30% identical), andaryl esterase hydrolase from Vibrio mimicus (27% identical). Based onincomplete sequence GDSL-type esterase (BRAIN) from Neisseriameningitidis (50% identical). Motifs: GDSL-GGND; no GRTT—replaced withGRTV. Sequence alignment against the core list of organisms shows theclosest association to Neisseria meningitidis, a member of thebeta-Proteobacteria.

M4aE11

Strongest hit: arylesterase from Agrobacterium tumefaciens (59%identity) Motifs: GDSL-GRTT-GTND. Sequence alignment against the corelist of organisms shows the closest association to members of thealpha-Proteobacteria such as Agrobacterium.

Est114

Strongest hit: phosphatidylcholine sterol acyltransferase from Aeromonashydrophila (gamma-Proteobacteria) (30% identical). Motifs: GDSL-GPND; noGRTT but GATT may be an equivalent. Sequence alignment against the corelist of organisms shows the closest association to Acidophilium sp. andAeromonas/Vibrio within the gamma-Proteobacteria.

Est105

Strongest hit: Pseudomonas aeruginosa outer membrane esterase, andhypothetical protein Pseudomonas putida (27% identical). Motifs:GDSL-GAND, no GRTT or equivalent. Sequence alignment against the corelist of organisms shows the closest association to members of thegamma-Proteobacteria.

An overall alignment of these clones/sequences (here shown underlined)indicates that they are scattered throughout the alignment tree ofstrains indicating that the metagenomic screening has provided a varietyof sequences and not a limited diversity.

Gene Mining for GRTT-Type Esterases

(clones with perhydrolase activity)

Sinorhizobium meliloti Sma1993-hypothetical protein_Sme

Motifs: GDSL-ARTT-GTND

Sinorhizobium meliloti Q92XZ1-hypothetical protein_Sme

Motifs: GDSN-GRTT-GTND

Mesorhizobium loti Q98MY5-arylesterase_Mlo

Motifs: GDSL-GRTT-GAND

Moraxella bovis AAK53448 (lipase)

Motifs: GDSL-GSND, no GRTT or equivalent in this sequence order.

(perhydrolase activity low, questionable sequence)

Agrobacterium tumefaciens Q8UACO

Motifs: GDSL-GRTT-GTND

Agrobacterium tumefaciens Q8UFG4

Motifs: GDSL-GRTT-GTND

Mesorhizobium loti RMLO00301

Motifs: GDSL-GRTT-GAND

Sinorhizobium meliloti RSM05666

Motifs: GDSL-GRTT-GSND

(this clone was inactive for perhydrolase activity;

and probably represents a false negative)

Sinorhizobium meliloti RSM02162

Motifs: GDSL-ARTT-GTND

Prosthecobacter dejongeii RVM05432

Motifs: GDSN-GRTT-GTND

A GDSx₁-x₂RTT-Gx₃ND motif characterizes the active clones/sequences,

where:

X₁=L or N

X₂=A or G

X₃=T or A or S

The Moraxella bovis AAK53448 sequence does not fit this pattern and isexcluded from the alignment analysis provided below:

Multiple Sequence Alignment of Active Clones/Sequences1                                               50 ACT MSMEG (1)-------------MAKRILCFGDSLUWGWVPVEDGAPU-ERFAPDVRWUG Q98MY5 Mesorhizobiumloti (1) --------------MKTVLCYGDSLTWGYNAEGGR------HALEDRWPS Sma1993Sinorhizobium (1) MTINSHSWRTLMVEKRSVLCFGDSLTWGWIPVKESSPT-LRYPYEQRWTGmeliloti Q92XZ1 Sinorhizobium meliloti (1)---------MEETVARTVLCFGDSNTHGQVPGRGPLDR---YRREQRWGG P. dejongeii RVM04532(1) --------------MKTILCFGDSNTWGYDPASMTAPFPRRHGPEVRWTG RSM05666_Sm (1)--------------MKTVLCYGDSLTWGYDATGSG-----RHALEDRWPS RSM02162_Sm (1)-----------MVEKRSVLCFGDSLTWGWIPVKESSPT-LRYPYEQRWTG At-Q8UAC0 (1)--------------MKTVLAFGDSLTWGADPATGLR-----HPVEHRWPD At-Q8UFG4 (1)-------------MVKSVLCFGDSLTWGSNAETGG-----RHSHDDLWPS M1-RML000301 (1)MAGGTRLDECTGERMKTVLCYGDSLTWGYNAEGGR------HALEDRWPS S261_M2aA12 (1)--------------MKNILAFGDSLTWGFVAGQDA-----RHPFETRWPN M091_M4aE11 (1)--------------MKTILAYGDSLTYGANPIPGG-PR---HAYEDRWPT Consensus (1)              MKTVLCFGDSLTWGY P  G      RHA E RWP51                                             100 ACT MSMEG (37)VLAQQLGADFEVIE--EGLSARUUNIDDPUDPRL-NGASYLPSCLAUHLP Q98MY5 Mesorhizobiumloti (31) VLQASLGGGVQVIA--DGLNGRTTAFDDHLAGADRNGARLLPTALTTHAP Sma1993Sinorhizobium (50) AMAARLGDGYHIIE--EGLSARTTSLDDPNDARL-NGSTYLPMALASHLPmeliloti Q92XZ1 Sinorhizobium meliloti (39)VLQGLLGPNWQVIE--EGLSGRTTVHDDPIEGSLKNGRIYLRPCLQSHAP P. dejongeii RVM04532(37) VLAKALGAGFRVIE--EGQNGRTTVHEDPLNICR-KGKDYLPACLESHKP RSM05666_Sm (32)VLQKALGSDAHVIA--EGLNGRTTAYDDHLADCDRNGARVLPTVLHTHAP RSM02162_Sm (39)AMAARLGDGYHIIE--EGLSARTTSLDDPNDARL-NGSTYLPMALASHLP At-Q8UAC0 (32)VLEAELAGKAKVHP--EGLGGRTTCYDDHAGPACRNGARALEVALSCHMP At-Q8UFG4 (33)VLQKALGSDVHVIFTHEGLGGRTTAYDDHTGDCDRNGARLLPTLLHSHAP M1-RMLO00301 (45)VLQASLGGGVQVIA--DGLNGRTTAFDDHLAGADRNGARLLPTALTTHAP S261_M2aA12 (32)ALAAGLGGKARVIE--EGQNGRTTVFDDAATFESRNGSVALPLLLISHQP M091_M4aE11 (33)ALEQGLGGKARVIA--EGLGGRTTVHDDWFANADRNGARVLPTLLESHSP Consensus (51) VL ALGG   VIE  EGL GRTTAHDD  A   RNGAR LPT L SHAP101                                            150 ACT MSMEG (84)LDLVIIMLGUNDUKAYFRRUPLDIA--LGMSVLVUQVLUSAGGVGUUYTA Q98MY5 Mesorhizobiumloti (79) IDLIVIMLGANDMKPWIHGNPVAAK--QGIQRLIDIVRGHDYPFDWPAP- Sma1993Sinorhizobium (97) LDLVIIMLGTNDTKSYFHRTPYEIA--NGMGKLVGQVLTCAGGVGTPYPAmeliloti Q92XZ1 Sinorhizobium meliloti (87)LDLIIIMLGTNDLKRRFNMPPSEVA--MGIGCLVHDIRELSPGRTGN--- P. dejongeii RVM04532(84) LDLVILMLGTNDLKSTFNVPPGEIA--AGAGVLGRMILAGDAGPENR-PP RSM05666_Sm (80)LDLIVFMLGSNDMKPIIHGTAFGAV--KGIERLVNLVRRHDWPTETEEG- RSM02162_Sm (86)LDLVIIMLGTNDTKSYFHRTPYEIA--NGMGKLVGQVLTCAGGVGTPYPA At-Q8UAC0 (80)LDLVIIMLGTNDIKPVHGGRAEAAVS--GMRRLAQIVETFIYKPREAVP- At-Q8UFG4 (83)LDMVIIMLGTNDMKPAIHGSAIVAFTMKGVERLVKLTRNHVWQVSDWEAP M1-RMLO00301 (93)IDLIVIMLGANDMKPWIHGNPVAAK--QGIQRLIDIVRGHDYPFDWPAP- S261_M2aA12 (80)LDLVIIMLGTNDIKFAARCRAFDAS--MGMERLIQIVRSANYMKGYKIP- M091_M4aE11 (81)LDLIVIMLGTNDIKPHHGRTAGEAG--RGMARLVQIIRGHYAGRMQDEP- Consensus (101)LDLVIIMLGTNDMKP  H  P EAA   GM RLV IVR   YG     P151                                            200 ACT MSMEG (132)PKVLVVSPPPLAPMPHPWFQLIFE--GGEQKUUELARVYSALASFMKVPF Q98MY5 Mesorhizobiumloti (126) -QILIVSPPVVSRTENADFREMFAG--GDEASKQLAPQYAALADEVGCGF Sma1993Sinorhizobium (145) PKVLWAPPPLAPMPDPWFEGMFG--GGYEKSKELSGLYKALADFMKVEFmeliloti Q92XZ1 Sinorhizobium meliloti (132)DPEIMIVAPPPMLEDLKEWESIFS--GAQEKSRKLALEFEIMADSLEAHF P. dejongeii RVM04532(131) QLLLMCPPKVRDLSAMPDLDAKIP--HGAARSAEFPRHYKAQAVALKCEY RSM05666_Sm(127) PEILIVSPPPLCETANSAFAAMFAG--GVEQSAMLAPLYRDLADELDCGF RSM02162_Sm(134) PKVLVVAPPPLAPMPDPWFEGMFG--GGYEKSKELSGLYKALADFMKVEF At-Q8UAC0 (127)-KLLIVAPPPCVAGPGGEPAGGRD----IEQSMRLAPLYRKLAAELGHHF At-Q8UFG4 (133)-DVLIVAPPQLCETANPFMGAIFRDAIDESAMLASVFTYRDLADELDCGF M1-RML000301 (140)-QILIVSPPVVSRTENADFREMFAG--GDEASKQLAPQYAALADEVGCGF S261_M2aA12 (127)-EILIISPPSLVPTQDEWFNDLWG--HAIAESKLFAKHYKRVAEELKVHF M091_M4aE11 (128)-QIILVSPPPIILGDWADMMDHFGPHEAIATSVDFAREYKKRADEQKVHF Consensus (151)  ILIVSPPPL  T   DF AMFG   G E SK LA  YKALADELK  F201                                   241 ACT MSMEG (180)FDAGSVISUDGVDGIHFUEANNRDLGVALAEQVRSLL---- (SEQ ID NO: 662) Q98MY5Mesorhizobium loti (173) FDAGTVAQTTPLDGVHLDAENTRNIGKALTSVVRVMLEL-- (SEQID NO: 663) Sma1993 Sinorhizobium (193)FAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF---- (SEQ ID NO: 664) melilotiQ92XZ1 Sinorhizobium meliloti (180)FDAGTVCQCSPADGFHIDEDAHRLLGEALAQEVLAIGWPDA (SEQ ID NO: 665) P. dejongeiiRVM04532 (179) FNSQEIVETSPVDGIHLEASEHLKLGEALAEKVKVLLG--- (SEQ ID NO:666) RSM05666_Sm (175) FDGGSVARTTPIDGVHLDAENTRAVGRGLEPVVRMMLGL-- (SEQ IDNO: 667) RSM02162_Sm (182) FAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF----(SEQ ID NO: 668) At-Q8UAC0 (172)FDAGSVASASPVDGVHLDASATAAIGRALAAPVRDILG--- (SEQ ID NO: 669) At-Q8UFG4(182) FDAGSVARTTPVDGVHLDAENTRAIGRGLEPVVRMMLGL-- (SEQ ID NO: 670)M1-RMLO00301 (187) FDAGTVAQTTPLDGVHLDAENTRNIGKALTSVVRVML---- (SEQ ID NO:671) S261_M2aA12 (174) FDAGTVAVADKTDGGHLDAVNTKAIGVALVPVVKSILAL-- (SEQ IDNO: 672) M091_M4aE11 (177) FDAGTVATTSKADGIHLDPANTRAIGAGLVPLVKQVLGL--(SEQ ID NO: 673) Consensus (201) FDAGTVA TSPVDGIHLDAENTR IG ALA VVR LLG(SEQ ID NO: 674)

A guide tree (i.e., an approximation of a phylogenetic tree) of theCLUSTALW alignment of active clones/sequences is provided below.

TABLE 13-2 Similarity and Identity of Clones/Sequences Compared to M.smegmatis Perhydrolase % % Clone/Sequence Identity SimilaritySinorhizobium meliloti Sma1993 55.5 71.6 Sinorhizobium meliloti Q92XZ138.7 54.7 Mesorhizobium loti Q98MY5 38.8 53.4 Moraxella bovis AAK534485.0 9.7 Agrobacterium tumefaciens Q8UACO 36.7 47.7 Agrobacteriumtumefaciens Q8UFG4 37.1 50.4 Mesorhizobium loti RMLO00301 34.8 50.9Sinorhizobium meliloti RSM05666 37.4 52.5 Sinorhizobium melilotiRSM02162 58.3 75.2 Prosthecobacter dejongeii RVM05432 41.6 55.7S261_M2aA12 39.3 54.3 M091_M4aE11 34.7 50.2

Based on the results, the active clones were found to have an overallidentity to M. smegmatis perhydrolase of 38.7-58.3%. Moraxella bovisAAK53448 was found to be an exception and the (translated) amino acidsequence is questionable.

Redundancy

From the analyses above, it was evident that some redundancy exists inthe alignment provided at the beginning of this Example that will haveadded undue weighting to the consensus sequence. Also, further GDSL-GRTTsequences were added. Thus, in the revised alignment below, thefollowing changes were made:

Removed:

Natural isolate 14B

Natural isolate 2D

RSM02162_Sm

Q98MY5 Mesorhizobium loti

Added:

BAB16197 (Arh II)

BAB16192 (Arh I)

NP 00197751 (Mlo II)

NP 00216984 (Bce)

NP 522806 (Rso)

Non-redundant alignment:

1                                               50 20A (1)-------------LPSGILCFGDSLTWGWIPVEEGVPTERFP-RDVRWTG 9B Natural Isolate(1) -GGRCVASCEVGAVAKRILCFGDSLTWGWIPVEEGVPTQRFP-KRVRWTG M. parafortuitumCO1 (1) -------------MAKRILCFGDSLTWGWIPVEEGVPTERFP-RDVRWTG MSAT (1)-------------MAKRILCFGDSLTWGWVPVEDGAPTERFA-PDVRWTG Sm-RSM05666 (1)--------------MKTVLCYGDSLTWGYDATG-----SGRHALEDRWPS At-Q8UAC0 (1)--------------MKTVLAFGDSLTWGADPAT-----GLRHPVEHRWPD At-Q8UFG4 (1)-------------MVKSVLCFGDSLTWGSNAET-----GGRHSHDDLWPS M091_M4aE11 (1)--------------MKTILAYGDSLTYGANPIP----GGPRHAYEDRWPT M1-RML000301 (1)MAGGTRLDECTGERMKTVLCYGDSLTWGYNAE------GGRHALEDRWPS P. dejongeii RVM04532(1) --------------MKTILCFGDSNTWGYDPASMTAPFPRRHGPEVRWTG Q92XZ1Sinorhizobium meliloti (1)---------MEETVARTVLCFGDSNTHGQVPG--RGPLDRYR-REQRWGG S261_M2aA12 (1)--------------MKNILAFGDSLTWGFVAG-----QDARHPFETRWPN Sma1993 Sinorhizobiummeliloti (1) MTINSHSWRTLMVEKRSVLCFGDSLTWGWIPVKESSPTLRYP-YEQRWTGZP_00197751 (1) --------------MKTILCYGDSLTWGYDAVG-----PSRHAYEDRWPSZP_00216984 (1) ----------MTMTQKTVLCYGDSNTHGTRPMTHAGGLGRFA-REERWTGBAB16192 (1) -----MICHKGGEEMRSVLCYGDSNTHGQIPG--GSPLDRYG-PNERWPG BAB16197(1) -----------MAESRSILCFGDSLTWGWIPVPESSPTLRYP-FEQRWTG NP_522806 (1)--------------MQQILLYSDSLSWGIIPG-----TRARLPFAARWAG Consensus (1)              MKTILCFGDSLTWGWIPV    P  RR   E RW G51                                             100 20A (37)VLADLLGDRYEVIE---EGLSARTTTADDPADPRLN-GSQYLPSCLASHL 9B Natural Isolate(49) VLADELGAGYEVVE---EGLSARTTTADDPTDPRLN-GSDYLPACLASHL M. parafortuitumCO1 (37) VLADLLGDRYEVIE---EGLSARTTTAEDPADPRLN-GSQYLPSCLASHL MSAT (37)VLAQQLGADFEVIE---EGLSARTTNIDDPTDPRLN-GASYLPSCLATHL Sm-RSM05666 (32)VLQKALGSDAHVIA---EGLNGRTTAYDDHLADCDRNGARVLPTVLHTHA At-Q8UAC0 (32)VLEAELAGKAKVHP---EGLGGRTTCYDDHAGPACANGARALEVALSCHM At-Q8UFG4 (33)VLQKALGSDVHVIFT-HEGLGGRTTAYDDHTGDCDRNGARLLPTLLHSHA M091_M4aE11 (33)ALEQGLGGKARVIA---EGLGGRTTVHDDWFANADRNGARVLPTLLESHS Ml-RML000301 (45)VLQASLGGGVQVIA---DGLNGRTTAFDDHLAGADANGARLLPTALTTHA P. dejongeii RVM04532(37) VLAKALGAGFRVIE---EGQNGRTTVHEDPLNICRK-GKDYLPACLESHK Q92XZ1Sinorhizobium meliloti (39)VLQGLLGPNWQVIE---EGLSGRTTVHDDPIEGSLKNGRIYLRPCLQSHA S261_M2aA12 (32)ALAAGLGGKARVIE---EGQNGRTTVFDDAATFESRNGSVALPLLLISHQ Sma1993 Sinorhizobiummeliloti (50) AMAARLGDGYHIIE---EGLSARTTSLDDPNDARLN-GSTYLPMALASHLZP_00197751 (32) VLQGRLGSSARVIA---EGLCGRTTAFDDWVAGADANGARILPTLLATHSZP_00216984 (40) VLAQTLGASWRVIE---EGLPARTTVHDDPIEGRHKNGLSYLRACVESHLBAB16192 (43) VLRRELGSQWYVIE---EGLSGRTTVRDDPIEGTMKNGRTYLRPCLMSHABAB16197 (39) AMAAALGDGYSIIE---EGLSARTTSVEDPNDPRLN-GSAYLPMALASHLNP_522806 (32) VMEHALQAQGHAVRIVEDCLNGRTTVLDDPARPGRN-GLQGLAQRIEAHAConsensus (51) VLA  LGA Y VIE   EGL GRIT  DDP D   RNGA YLP  L SH101                                            150 20A (83)PLDLVILMLGINDTKANFGRTPFD--IATGMGVLATQVLTSAGG-VGTSY 9B Natural Isolate(95) PLDLVILMLGTNDTKANLNRTPVD--IASGMGVLATQVLTSAGG-VGTSY M. parafortuitumCO1 (83) PLDLVILMLGTNDTKANFGRTPFD--IATGMGVLATQVLTSAGG-VGTSY MSAT (83)PLDLVIIMLGTNDTKAYFRRTPLD--IALGMSVLVTQVLTSAGG-VGTTY Sm-RSM05666 (79)PLDLIVFMLGSNDMKPIIHGTAFG--AVKGIERLVNLVRRHDWPT--ETE At-Q8UAC0 (79)PLDLVIIMLGTNDIKPVHGGRAEA--AVSGMARLAQIVETFIYK---PRE At-Q8UFG4 (82)PLDMVIIMLGTNDMKPAIHGSAIVAFTMKGVERLVKLTRNHVWQV--SDW M091_M4aE11 (80)PLDLIVIMLGTNDIKPHHGRTAGE--AGRGMARLVQIIRGHYAG---RMQ Ml-RML000301 (92)PIDLIVIMLGANDMKPWIHGNPVA--AKQGIQRLIDIVRGHDYP---FDW P. dejongeii RVM04532(83) PLDLVILMLGTNDLKSTFNVPPGE--IAAGAGVLGRMILAGDA---GPEN Q92XZ1Sinorhizobium meliloti (86)PLDLIIIMLGTNDLKRRFNMPPSE--VAMGIGCLVHDIRELSP---GRTG S261_M2aA12 (79)PLDLVIIMLGTNDIKFAARCRAFD--ASMGMERLIQIVRSANYM---KGY Sma1993 Sinorhizobiummeliloti (96) PLDLVIIMLGTNDTKSYFHRTPYE--IANGMGKLVGQVLTCAGG-VGTPYZP_00197751 (79) PLDLVIVMLGTNDMKSFVCGRAIG--AKQGMERIVQIIRGQPYS---FNYZP_00216984 (87) PVDVVVLMLGTNDLKTRFSVTPAD--IATSVGVLLAKIAACGA---GPSGBAB16192 (90) ILDLVIIMLGTNDLKARFGQPPSE--VAMGIGCLVYDIRELAP---GPGGBAB16197 (85) PLDLVIILLGTNDTKSYFRRTPYE--IANGMGKLAGQVLTSAGG-IGTPYNP_522806 (81) PLALVILMLGTNDFQAIFRHTAQD--AAQGVAQLVRAIRQAPIEP---GMConsensus (101) PLDLVIIMLGTNDLKA F  TP D  IA GMGRLV  VR   G   G  Y151                                            200 20A (130)PAPQVLIVAPPPLGELPHPWFDL--VFSGGREKTAELARVYSALASFMKV 9B Natural Isolate(142) PAPQVLIVAPPPLAEMPHPWFEL--VFDGGREKTAQLARVYSALASFMKV M.parafortuitum CO1 (130)PAPQVLIVAPPPLGELPHPWFDL--VFSGGREKTAELARVYSALASFMKV MSAT (130)PAPKVLVVSPPPLAPMPHPWFQL--IFEGGEQKTTELARVYSALASFMKV Sm-RSM05666 (125)EGPEILIVSPPPLCETANSAFAAMFAGGVEQSAMLAP--LYRDLADELDC At-Q8UAC0 (124)AVPKLLIVAPPPCVAGP--GGEPAGGRDIEQSMRLAP--LYRKLAAELGH At-Q8UFG4 (130)EAPDVLIVAPPQLCETANPFMGAIFRDAIDESAMLASVFTYRDLADELDC M091_M4aE11 (125)DEPQIILVSPPPIILGDWADMMDHFGPHEAIATSVDFAREYKKRADEQKV Ml-RML000301 (137)PAPQILIVSPPVVSRTENADFREMFAGGDEASKQLAP--QYAALADEVGC P. dejongeii RVM04532(128) RPPQLLLMCPPKVRDLSAMPDLDAKIPHGAAR-SAEFPRHYKAQAVALKC Q92XZ1Sinorhizobium meliloti (131)NDPEIMIVAPPPMLEDLKEWES---IFSGAQEKSRKLALEFEIMADSLEA S261_M2aA12 (124)KIPEILIISPPSLVPTQDEWFNDLWGHAIAESKLFAK--HYKRVAEELKV Sma1993 Sinorhizobiummeliloti (143) PAPKVLVVAPPPLAPMPDPWFEG--MFGGGYEKSKELSGLYKALADFMKVZP_00197751 (124) KVPSILLVAPPPLCATENSDFAEIFEGGMAESQKLAP--LYAALAQQTGCZP_00216984 (132) ASPKLVLMAPAPIVEVGFLGEI---FAGGAAK-SRQLAKRYEQVASDAGABAB16192 (135) KPPEIMVVAPPPMLDDIKEWEP---IFSGAQEKSRRLALEFEIIADSLEVBAB16197 (132) PAPKLLIVSPPPLAPMPDPWFEG--MFGGGYEKSLELAKQYKALANFLKVNP_522806 (126) PVPPVLIVVPPAITAPAGAMADK---FADAQPKCAGLAQAYRATAQTLGCConsensus (151)  AP ILIVAPPPL E    WF    IFGGA  KS  LA  YKALA  LKV201                                            248 20A (178)PFFDAGSVISTDGVDGTHFTRGETI----------------------- (SEQ ID NO: 675) 9BNatural Isolate (190) PFFDAGSVISTDGVDGTHFTRGETIDR---------------------(SEQ ID NO: 676) M. parafortuitum CO1 (178)PFFDAGSVISTDGVDGIHFTRGEQST---------------------- (SEQ ID NO: 677) MSAT(178) PFFDAGSVISTDGVDGIHFTEANNRDLGVALAEQVRSLL--------- (SEQ ID NO: 678)Sm-RSM05666 (173) GFFDGGSVARTTPIDGVHLDAENTRAVGRGLEPVVRMMLGL------- (SEQID NO: 679) At-Q8UAC0 (170)HFFDAGSVASASPVDGVHLDASATAAIGRALAAPVRDILG-------- (SEQ ID NO: 680)At-Q8UFG4 (180) GFFDAGSVARTTPVDGVHLDAENTRAIGRGLEPVVRMMLGL------- (SEQ IDNO: 681) M091_M4aE11 (175)HFFDAGTVATTSKADGIHLDPANTRAIGAGLVPLVKQVLGL------- (SEQ ID NO: 682)Ml-RML000301 (185) GFFDAGTVAQTTPLDGVHLDAENTRNIGKALTSVVRVML--------- (SEQID NO: 683) P. dejongeii RVM04532 (177)EYFFSQEIVETSPVDGIHLEASEHLKLGEALAEKVKVLLG-------- (SEQ ID NO: 684) Q92XZ1Sinorhizobium meliloti (178)HFFDAGTVCQCSPADGFHIDEDAHRLLGEALAQEVLAIGWPDA----- (SEQ ID NO: 685)S261_M2aA12 (172) HFFDAGTVAVADKTDGGHLDAVNTKAIGVALVPVVKSILAL------- (SEQID NO: 686) Sma1993 Sinorhizobium meliloti (191)EFFAAGDCISTDGIDGIHLSAETNIRLGHAIADKVAALF--------- (SEQ ID NO: 687ZP_00197751 (172) AFFDAGTVARTTPLDGIHLDAENTRAIGAGLEPVVRQALGL------- (SEQID NO: 688) ZP_00216984 (178)HFLDAGAIVEVSPVDGVHFAADQHRVLGQRVAALLQQIA--------- (SEQ ID NO: 689)BAB16192 (182) HFFDAATVASCDPCDGFHINREAHEALGTALAREVEAIGWR------- (SEQ IDNO: 690) BAB16197 (180) DFLDAGEFVKIDGCDGIHFSAETNITLGHAIAAKVEAIFSQEAKNAAA(SEQ ID NO: 691) NP_522806 (173)HVFDANSVTPASAVDGIHLDADQHAQLGRAMAQVVGTLLAQ------- (SEQ ID NO: 692)Consensus (201)  FFDAGSV  TSPVDGIHLDAENTR LG ALA  VR IL (SEQ ID NO: 693)

The guide tree to the CLUSTALW alignment (which approximates to aphylogenetic tree) clearly indicates 3 groupings:

1) GDSL-ARTT group including Act

2) GDSL-GRTT group composed of members of the Rhizobiales and themetagenome; and

3) Intermediate group of mixed motifs.

It is also contemplated that the results suggest some form of geneduplication and mutation events in the Rhizobiales and lateral genetransfer to Mycobacterium.

Using the non-redundant alignment a new Act consensus was constructedcalled “Act chimera”.

(SEQ ID NO: 694) 1 KTILCFGDSL TWGWIPVEDG APTERRAPEV RWTGVLAQQLGADYEVIEEG 51 LSGRTTNIDD PTDPRLRNGA SYLPSCLASH LPLDLVIIML GTNDLKAYFR 101RTPLDIALGM GRLVTQVRTS AGGVGTTYPA PKILIVAPPP LAEMPHPWFQ 151 LIFGGAEQKSTELARVYKAL ASFLKVPFFD AGSVISTSPV DGIHLDAENT 201 RDLGVALAEQ VRSIL

An alignment of Act-chimera with Ms Act (Chimera align) indicates 91.6%similarity and 86.0% identity, as indicated below.

1                                               50 MSAT (1)MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIE Act-Chimera (1)--KTILCFGDSLTWGWIPVEDGAPTERRAPEVRWTGVLAQQLGADYEVIE Consensus (1)   KILCFGDSLTWGWIPVEDGAPTER APDVRWTGVLAQQLGADFEVIE51                                             100 MSAT (51)EGLSARTTNIDDPTDPRLN-GASYLPSCLATHLPLDLVIIMLGTNDTKAY Act-Chimera (49)EGLSGRTTNIDDPTDPRLRNGASYLPSCLASHLPLDLVIIMLGTNDLKAY Consensus (51)EGLSARTTNIDDPTDPRL  GASYLPSCLASHLPLDLVIIMLGTND KAY101                                            150 MSAT (100)FRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPHPW Act-Chimera (99)FRRTPLDIALGMGRLVTQVRTSAGGVGTTYPAPKILIVAPPPLAEMPHPW Consensus (101)FRRTPLDIALGM  LVTQV TSAGGVGTTYPAPKILIVAPPPLA MPHPW151                                            200 MSAT (150)FQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEA Act-Chimera (149)FQLIFGGAEQKSTELARVYKALASFLKVPFFDAGSVISTSPVDGIHLDAE Consensus (151) FQLIFGAEQKSTELARVY ALASFLKVPFFDAGSVIST  VDGIH 201           217 MSAT (200)NNRDLGVALAEQVRSLL  (SEQ ID NO: 695) Act-Chimera (199)NTRDLGVALAEQVRSIL  (SEQ ID NO: 694) Consensus (201) NRDLGVALAEQVRSIL  (SEQ ID NO: 696)

A BLASTP search with Act-chimera did not reveal any further sequences.

The Act-chimera is “forced” on the Per sequence at the positions whereno consensus exists. However, a basic ‘unforced’ consensus sequence didnot provide any more information from a blastp search or from alignmentanalysis. Thus, comparison with the most distant homologues in theblastp ‘hit’ list was considered more useful in defining the importantresidues/positions in Act sequence space. This was a useful exercise, asthese sequences were not used in the non-redundant alignment.

For example, Rhodopirellula baltica (NP_(—)865748; Psp; a Planctomycetesand quite different from either Mycobacterium or Rhizobiales), wascompared as shown below.

1                                               50 MSAT (1)MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLA---QQLGADFE NP_865746 (1)-MHSILIYGDSLSWGIIPGTR----RRFAFHQRWPGVMEIELRQTGIDAR Consensus (1)     ILFGDSLSWG IP      RFA   RW GVL     Q G D51                                             100 MSAT (48)VIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTK NP_865746 (46)VIEDCLNGRRTVLEDPIKPGRNGLDGLQQRIEINSPLSLVVLFLGTNDFQ Consensus (51) VIED LAR T IDDP  P  NG   L   I    PL LVII LGTND101                                            150 MSAT (98)AYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPH NP_865746 (96)SVHEFHAEQSAQGLALLV--DAIRRSPFEPGMPTPKILLVAPPTVHH-PK Consensus (101)A        A GLALLV              P PKILLVAPP L   P151                                            200 MSAT (148)PWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFT NP_865746 (143)LDMAAKFQNAETKSTGLADAIRKVSTEHSCEFFDAATVTTTSVVDGVHLD Consensus (151)      F  AE KST LA     LAS     FFDAASV ST  VDGIH 201                222MSAT (198) EANNRDLGVALAEQVRSLL---  (SEQ ID NO: 695) NP_865746 (193)QEQHQALGTALASTIAEILADC  (SEQ ID NO: 697) Consensus (201)   N   LGALA  I  IL  (SEQ ID NO: 698)The following is an alignment with Ralstonia eutropha (Reu):

1                                               50 MSAT (1)---------MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLA-- ZP_00166901 (1)MPLTAPSEVDPLQILVYADSLSWGIVPGTR----RRLPFPVRWPGRLELG Consensus (1)             IL FADSLSWG VP        R    VRW G L51                                             100 MSAT (40)--QQLGADFEVIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLV ZP_00166901 (47)LNADGGAPVRIIEDCLNGRRTVWDDPFKPGRNGLQGLAQRIEIHSPVALV Consensus (51)     GA   IIED L AR T  DDP  P  NG   L   I  H PL LV101                                            150 MSAT (88)IIMLGTNDTKAYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVV ZP_00166901 (97)VLMLGNNDFQSMHPHNAWHAAQGVGALV--HAIRTAPIEPGMPVPPILVV Consensus (101) IIMLGND  A         A GM  LV       A I    P P ILVV151                                            200 MSAT (138)SPPPLAPMPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVIS ZP_00166901 (145)VPPPIRT-PCGPLAPKFAGGEHKWAGLPEALRELCATVDCSLFDAGTVIQ Consensus (151)PPPI   P       F GGE K   L      L A M    FDAGSVI201                               237 MSAT (188)TDGVDGIHFTEANNRDLGVALAEQVRSLL-------- (SEQ ID NO: 695) ZP_00166901 (194)SSAVDGVHLDADAHVALGDALQPVVRALLAESSGHPS (SEQ ID NO: 699) Consensus (201) SAVDGIH        LG AL   VRALL (SEQ ID NO: 700)

Based on these results, the following conclusions were made. A BLASTpnr-database search with a perhydrolase consensus sequence revealed GDSLor GDSI lipases/esterases from a wide diversity of organisms. However,only 12 or 14 of these were reliable homologues of Per. Nearly all ofthese were derived from 1 small group of bacteria, namely theRhizobiales (i.e., Gram-negative soil bacteria belonging thealpha-Proteobacteria). A few members of the beta-Proteobacteria werefound, but no Mycobacterium sp. This provides an indication that theperhydrolase (Per) gene/protein is not widely distributed in nature.

The Mycobacterium protein is characterized by the GDSL-ARTT motif,whereas most of the Rhizobiales are characterized by a GDSL-GRTT motif.There are also some mixed or intermediate motifs (e.g., GDSN-GRTT,GDSN-ARTT and SDSL-GRTT). This may indicate gene duplication andmutation event and lateral gene transfer. The consensus residuesidentified in these experiments were L6, W14, R27, W34, L38, R56, D62,L74, L78, H81, P83, M90, K97, G110, L114, L135, F180, and G205.

Using the non-redundant alignment and comparison with distant homologuesthe follow sequence space can be defined starting at position 5 of theM. smegmatis perhydrolase and ending at position 195, with perhydrolaseshown in residues in bold. [I, V][L][X][F, Y][G, S][D][S][L, N][T, S][W,Y, H][G][X]₂[P, A][X]₁₄[R, L][W][X]₇[L][X]₅[V, I][I, V, H][X][E. D][G,C][L, Q][X][G, A][R][T][T][X]₂[D, E][D][X]₇[G][X]₃[L][X]₆[H][X][P, I][L,I, V][D, A][V, I][X]₂[M, L][L][G][X][N][D][X]₃₆[P][X]₆[P][P,A][X]₃₁[A][X]₁₉[D][G][X][H] (SEQ ID NO:701)

In sum, it is clear from the analyses above that the activeclones/sequences with a GDSx₁-x₂RTT-Gx₃ND motif have all been foundamong the alpha-Proteobacteria-Gram-negative bacteria associated withthe soil rhizosphere. This is in sharp contrast to the prototypeperhydrolase from M. smegmatis a high GC content Gram-positive bacteriumassigned to the class Actinobacteria. This division is illustrated inFIG. 2, which provides a phylogenetic tree, showing the major branchesof the bacteria and the origin of the active clones/sequences comparedto M. smegmatis.

Example 14 Native Molecular Weight Estimation of Homologues of thePerhydrolase

In this Example, experiments conducted to estimate the native molecularweights of M. smegmatis perhydrolase homologues are described.

Preparation of Samples for Purification (Size Determination)

A single colony of the desired strains was inoculated in 50 ml TerrificBroth and incubated overnight at 37° C. with shaking at 200 rpm. Thecells were pelleted by centrifugation for 10 minutes at 7000 rpm in aSorvall SuperSpeed Centrifuge. The pellets were then resuspended in 10ml 25 mM Bis-Tris (pH 6.5) and lysed by passage through a Frenchpressure cell twice. The lysates were then centrifuged at 15000 rpm in aSorvall SuperSpeed Centrifuge. The soluble fraction was heat treated at55° C. for 1 hour to precipitate cellular proteins. The samples werethen centrifuged at 10000 rpm in a Sorvall SuperSpeed Centrifuge and thesoluble fractions used for further purification or assay.

Sizing Columns

The supernatants (prepared as described above) were run on a Sephadex200 sizing column in 20 mM phosphate (pH 8.0), with a flow rate of 0.5ml/min. The column was calibrated prior to running the samples with MWstandards (listed below) and purified M. smegmatis perhydrolase protein.The crude sample elution volumes were determined by collecting 0.5 mlfractions, and assaying the fractions for pNB activity. Molecularweights and elution volumes of the standards:

Thyroglobulin MW 669 kDa: elution volume 16 ml

Aldolase MW 158 kDa: elution volume 24 ml

Ovalbumin MW 43 kDa: elution volume 26 ml

Ribonuclease MW 14 kDa: elution volume 32 ml

Perhydrolase elution volume 24 ml

Results

The following Table (Table 14-1) provides the elution volume of some ofthe M. smegmatis perhydrolase homologues identified herein.

TABLE 14-1 Elution Volume (Estimated Molecular Weight) of M. smegmatisPerhydrolase Homologues Homologue Sample Elution Volume (ml) pLO_SmeI 24pET26_SmeII 24 pET26_MIO 24 pET26b_Stm 24 pET26b_Mbo 24 M7OaEB_pET26 32pET26_m2aA12 24 pET26b_S2487am 32 S. meliloti RSM02162 (G00355) 24PET_M2aAl2 (5261) 24 M. smegmatis Perhydrolase 24

The data in the above Table and the assay results obtained for thesehomologues indicated that these enzymes have an amino acid sequencesimilar to the M. smegmatis perhydrolase. As with the M. smegmatisperhydrolase, these homologues exhibit perhydrolysis activity asmultimers. As described herein, the perhydrolase is an octamer, whilethe homologues, although they elute in a similar volume, arecontemplated to be dimers, trimers, tetramers, hexamers, and/oroctamers.

Example 15 Crystal Structure of Perhydrolase

In this Example, the crystallographic analysis of the perhydrolase isdescribed. Perhydrolase crystals were obtained under two conditions: 2.0M [NH₄]₂SO₄, 2% PEG400, 0.1 M Tris pH 7.1 (giving triclinic, P1crystals) and 1.0 M ammonium dihydrogen phosphate, and 0.1M sodiumcitrate pH 5.6 (giving tetragonal, P4 crystals) Both crystal forms gavesuitable diffraction beyond 2.0 Å resolution. Derivative protein for aMAD phase determination using selenium replacing sulfur containingmethionine resulting in a protein molecule having four selenomethioninesthe N-terminal methionine is cleaved proteolytically. Of the two forms,triclininc P1 a=83.77 Å b=90.07 Å c=112.115 Å α=73.32° β, 77.30°γ=88.07° and P4 a=b=98.18 Å c=230.12 Å, the P4 crystal gave data thatwas possible to use for structure determination. Three wavelength MADdatasets were collected at wavelengths corresponding to the Seabsorption edge, near the inflection point and a third, away from theabsorption edge.

Three hundred and thirty-three frames (0.3 degree oscillations perframe) for each wavelength with 1 sec exposure time were collected froma single tetragonal space group P4 crystal. The structure could besolved with either SOLVE or SHELX computer programs giving similarsolutions for the 32 possible Se positions. The map was fitted using theprogram “O”. It was possible to trace electron density for residues3-216 in each of the eight independent molecules. The final structure ofthese eight molecules was refined using CNS. The currentcrystallographic R-factor is 21%. The coordinates are provided below.

CRYST1 98.184 98.184 230.119 90.00 90.00 90.00 SCALE1 0.010185 0.0000000.000000 0.000000 SCALE2 0.000000 0.010185 0.000000 0.000000 SCALE30.000000 0.000000 0.004346 0.000000 ATOM 1 CB LYS 3 −8.167 −61.96418.588 1.000 40.95 ATOM 2 CG LYS 3 −8.685 −63.192 19.323 1.000 22.95ATOM 3 CD LYS 3 −8.635 −64.400 18.399 1.000 14.97 ATOM 4 CE LYS 3 −7.963−65.575 19.090 1.000 19.83 ATOM 5 NZ LYS 3 −7.359 −66.511 18.099 1.00044.28 ATOM 6 C LYS 3 −9.684 −60.377 17.426 1.000 13.89 ATOM 7 O LYS 3−9.087 −59.356 17.767 1.000 12.50 ATOM 8 N LYS 3 −8.000 −61.626 16.1531.000 15.57 ATOM 9 CA LYS 3 −8.919 −61.686 17.284 1.000 20.71 ATOM 10 NARG 4 −10.987 −60.381 17.166 1.000 24.56 ATOM 11 CA ARG 4 −11.695−59.097 17.204 1.000 22.65 ATOM 12 CB ARG 4 −12.299 −58.822 15.822 1.00021.44 ATOM 13 CG ARG 4 −11.232 −58.465 14.792 1.000 21.56 ATOM 14 CD ARG4 −11.845 −58.181 13.431 1.000 29.29 ATOM 15 NE ARG 4 −11.660 −56.79013.020 1.000 32.87 ATOM 16 CZ ARG 4 −12.643 −56.013 12.585 1.000 30.24ATOM 17 NH1 ARG 4 −13.879 −56.487 12.494 1.000 17.82 ATOM 18 NH2 ARG 4−12.399 −54.760 12.229 1.000 44.53 ATOM 19 C ARG 4 −12.735 −59.05418.308 1.000 14.59 ATOM 20 O ARG 4 −13.604 −59.909 18.456 1.000 18.72ATOM 21 N ILE 5 −12.639 −58.012 19.131 1.000 13.45 ATOM 22 CA ILE 5−13.549 −57.882 20.263 1.000 12.08 ATOM 23 CB ILE 5 −12.747 −57.83521.578 1.000 15.40 ATOM 24 CG2 ILE 5 −13.678 −57.677 22.765 1.000 5.80ATOM 25 CG1 ILE 5 −11.811 −59.034 21.741 1.000 11.66 ATOM 26 CD1 ILE 5−10.437 −58.632 22.232 1.000 19.35 ATOM 27 C ILE 5 −14.420 −56.64020.142 1.000 8.96 ATOM 28 O ILE 5 −13.905 −55.529 20.021 1.000 13.31ATOM 29 N LEU 6 −15.736 −56.833 20.169 1.000 13.04 ATOM 30 CA LEU 6−16.675 −55.728 20.059 1.000 8.54 ATOM 31 CB LEU 6 −17.879 −56.08719.178 1.000 7.42 ATOM 32 CG LEU 6 −18.959 −54.996 19.120 1.000 14.12ATOM 33 CD1 LEU 6 −18.446 −53.783 18.359 1.000 12.19 ATOM 34 CD2 LEU 6−20.245 −55.512 18.494 1.000 27.94 ATOM 35 C LEU 6 −17.170 −55.29321.436 1.000 2.72 ATOM 36 O LEU 6 −17.719 −56.101 22.179 1.000 13.36ATOM 37 N CYS 7 −16.978 −54.020 21.756 1.000 1.38 ATOM 38 CA CYS 7−17.472 −53.469 23.011 1.000 3.17 ATOM 39 CB CYS 7 −16.411 −52.58223.667 1.000 7.01 ATOM 40 SG CYS 7 −14.867 −53.471 23.992 1.000 11.21ATOM 41 C CYS 7 −18.755 −52.685 22.776 1.000 0.65 ATOM 42 O CYS 7−18.756 −51.627 22.145 1.000 4.76 ATOM 43 N PHE 8 −19.859 −53.228 23.2811.000 0.00 ATOM 44 CA PHE 8 −21.147 −52.568 23.053 1.000 1.14 ATOM 45 CBPHE 8 −22.115 −53.578 22.443 1.000 5.54 ATOM 46 CG PHE 8 −23.421 −53.00021.937 1.000 3.36 ATOM 47 CD1 PHE 8 −23.456 −52.212 20.800 1.000 0.89ATOM 48 CD2 PHE 8 −24.602 −53.262 22.614 1.000 1.39 ATOM 49 CE1 PHE 8−24.644 −51.683 20.333 1.000 0.00 ATOM 50 CE2 PHE 8 −25.793 −52.73322.148 1.000 4.42 ATOM 51 CZ PHE 8 −25.818 −51.944 21.012 1.000 2.71ATOM 52 C PHE 8 −21.677 −51.978 24.346 1.000 4.46 ATOM 53 O PHE 8−21.873 −52.672 25.348 1.000 6.98 ATOM 54 N GLY 9 −21.923 −50.666 24.3841.000 5.61 ATOM 55 CA GLY 9 −22.396 −50.109 25.646 1.000 5.44 ATOM 56 CGLY 9 −22.860 −48.673 25.522 1.000 5.66 ATOM 57 O GLY 9 −23.229 −48.22224.440 1.000 14.54 ATOM 58 N ASP 10 −22.837 −47.964 26.641 1.000 3.89ATOM 59 CA ASP 10 −23.322 −46.596 26.734 1.000 5.17 ATOM 60 CB ASP 10−24.331 −46.467 27.880 1.000 2.99 ATOM 61 CG ASP 10 −23.807 −47.05229.175 1.000 7.05 ATOM 62 OD1 ASP 10 −22.617 −46.829 29.494 1.000 17.93ATOM 63 OD2 ASP 10 −24.564 −47.738 29.895 1.000 10.98 ATOM 64 C ASP 10−22.154 −45.642 26.939 1.000 5.15 ATOM 65 O ASP 10 −21.022 −45.94026.556 1.000 5.62 ATOM 66 N SER 11 −22.423 −44.497 27.554 1.000 9.02ATOM 67 CA SER 11 −21.394 −43.493 27.802 1.000 3.43 ATOM 68 CB SER 11−22.014 −42.331 28.585 1.000 7.25 ATOM 69 OG SER 11 −22.640 −42.81329.763 1.000 18.93 ATOM 70 C SER 11 −20.199 −44.046 28.561 1.000 7.58ATOM 71 O SER 11 −19.089 −43.508 28.501 1.000 16.71 ATOM 72 N LEU 12−20.393 −45.133 29.308 1.000 6.56 ATOM 73 CA LEU 12 −19.264 −45.69630.046 1.000 16.41 ATOM 74 CB LEU 12 −19.711 −46.759 31.042 1.000 17.05ATOM 75 CG LEU 12 −20.598 −46.336 32.210 1.000 18.22 ATOM 76 CD1 LEU 12−20.866 −47.527 33.123 1.000 7.48 ATOM 77 CD2 LEU 12 −19.973 −45.18432.988 1.000 10.83 ATOM 78 C LEU 12 −18.269 −46.285 29.048 1.000 14.99ATOM 79 O LEU 12 −17.065 −46.307 29.267 1.000 6.10 ATOM 80 N THR 13−18.828 −46.764 27.940 1.000 14.77 ATOM 81 CA THR 13 −18.014 −47.34726.876 1.000 8.83 ATOM 82 CB THR 13 −18.828 −48.381 26.080 1.000 6.87ATOM 83 OG1 THR 13 −19.109 −49.487 26.949 1.000 10.08 ATOM 84 CG2 THR 13−18.033 −48.940 24.914 1.000 16.85 ATOM 85 C THR 13 −17.490 −46.24525.970 1.000 4.56 ATOM 86 O THR 13 −16.315 −46.220 25.616 1.000 11.71ATOM 87 N TRP 14 −18.376 −45.317 25.612 1.000 5.57 ATOM 88 CA TRP 14−17.992 −44.210 24.742 1.000 7.21 ATOM 89 CB TRP 14 −19.208 −43.32924.453 1.000 6.90 ATOM 90 CG TRP 14 −18.917 −42.183 23.537 1.000 11.88ATOM 91 CD2 TRP 14 −18.731 −40.813 23.924 1.000 13.72 ATOM 92 CE2 TRP 14−18.483 −40.081 22.745 1.000 11.95 ATOM 93 CE3 TRP 14 −18.752 −40.14725.152 1.000 10.63 ATOM 94 CD1 TRP 14 −18.779 −42.222 22.181 1.000 8.28ATOM 95 NE1 TRP 14 −18.517 −40.963 21.694 1.000 7.16 ATOM 96 CZ2 TRP 14−18.255 −38.705 22.763 1.000 5.39 ATOM 97 CZ3 TRP 14 −18.526 −38.78325.168 1.000 12.55 ATOM 98 CH2 TRP 14 −18.282 −38.084 23.981 1.000 12.81ATOM 99 C TRP 14 −16.880 −43.353 25.327 1.000 5.41 ATOM 100 O TRP 14−16.107 −42.745 24.582 1.000 4.90 ATOM 101 N GLY 15 −16.794 −43.28326.652 1.000 8.94 ATOM 102 CA GLY 15 −15.794 −42.475 27.318 1.000 4.51ATOM 103 C GLY 15 −16.249 −41.098 27.755 1.000 10.98 ATOM 104 O GLY 15−15.480 −40.136 27.646 1.000 15.11 ATOM 105 N TRP 16 −17.471 −40.95228.255 1.000 23.34 ATOM 106 CA TRP 16 −17.988 −39.691 28.792 1.000 15.10ATOM 107 CB TRP 16 −19.408 −39.890 29.327 1.000 6.11 ATOM 108 CG TRP 16−20.139 −38.694 29.846 1.000 1.78 ATOM 109 CD2 TRP 16 −21.229 −38.00829.213 1.000 8.98 ATOM 110 CE2 TRP 16 −21.613 −36.942 30.051 1.000 7.76ATOM 111 CE3 TRP 16 −21.923 −38.186 28.009 1.000 15.66 ATOM 112 CD1 TRP16 −19.927 −38.021 31.016 1.000 0.35 ATOM 113 NE1 TRP 16 −20.798 −36.97331.154 1.000 8.35 ATOM 114 CZ2 TRP 16 −22.649 −36.063 29.734 1.000 5.16ATOM 115 CZ3 TRP 16 −22.952 −37.317 27.692 1.000 5.34 ATOM 116 CH2 TRP16 −23.306 −36.269 28.551 1.000 4.72 ATOM 117 C TRP 16 −17.059 −39.15429.881 1.000 7.85 ATOM 118 O TRP 16 −16.846 −39.815 30.899 1.000 3.97ATOM 119 N VAL 17 −16.533 −37.952 29.685 1.000 5.45 ATOM 120 CA VAL 17−15.750 −37.256 30.695 1.000 12.08 ATOM 121 CB VAL 17 −14.822 −36.19130.082 1.000 17.55 ATOM 122 CG1 VAL 17 −14.084 −35.443 31.185 1.00011.59 ATOM 123 CG2 VAL 17 −13.841 −36.807 29.099 1.000 7.77 ATOM 124 CVAL 17 −16.673 −36.565 31.696 1.000 13.86 ATOM 125 O VAL 17 −17.390−35.618 31.351 1.000 1.02 ATOM 126 N PRO 18 −16.660 −37.034 32.936 1.0008.38 ATOM 127 CD PRO 18 −15.770 −38.071 33.476 1.000 8.64 ATOM 128 CAPRO 18 −17.572 −36.501 33.948 1.000 9.99 ATOM 129 CB PRO 18 −17.201−37.294 35.208 1.000 12.31 ATOM 130 CG PRO 18 −15.817 −37.789 34.9541.000 7.46 ATOM 131 C PRO 18 −17.327 −35.017 34.191 1.000 13.05 ATOM 132O PRO 18 −16.163 −34.619 34.306 1.000 18.63 ATOM 133 N VAL 19 −18.381−34.211 34.266 1.000 6.92 ATOM 134 CA VAL 19 −18.214 −32.793 34.5851.000 9.29 ATOM 135 CB VAL 19 −18.482 −31.856 33.388 1.000 5.33 ATOM 136CG1 VAL 19 −17.377 −31.995 32.354 1.000 6.78 ATOM 137 CG2 VAL 19 −19.850−32.150 32.796 1.000 3.72 ATOM 138 C VAL 19 −19.151 −32.380 35.710 1.00012.02 ATOM 139 O VAL 19 −20.217 −32.962 35.913 1.000 14.52 ATOM 140 NGLU 20 −18.771 −31.351 36.467 1.000 17.17 ATOM 141 CA GLU 20 −19.662−30.994 37.575 1.000 13.30 ATOM 142 CB GLU 20 −18.918 −30.130 38.5951.000 25.34 ATOM 143 CG GLU 20 −18.276 −30.968 39.702 1.000 31.46 ATOM144 CD GLU 20 −16.871 −30.487 40.017 1.000 35.91 ATOM 145 OE1 GLU 20−16.143 −30.157 39.055 1.000 40.11 ATOM 146 OE2 GLU 20 −16.507 −30.43141.210 1.000 45.47 ATOM 147 C GLU 20 −20.913 −30.294 37.080 1.000 7.56ATOM 148 O GLU 20 −21.964 −30.361 37.723 1.000 11.30 ATOM 149 N ASP 21−20.852 −29.610 35.936 1.000 19.38 ATOM 150 CA ASP 21 −22.099 −28.98335.471 1.000 23.47 ATOM 151 CB ASP 21 −21.815 −27.740 34.640 1.000 17.53ATOM 152 CG ASP 21 −21.114 −27.991 33.326 1.000 14.93 ATOM 153 OD1 ASP21 −20.984 −29.159 32.908 1.000 26.78 ATOM 154 OD2 ASP 21 −20.685−26.996 32.694 1.000 8.74 ATOM 155 C ASP 21 −22.959 −29.988 34.707 1.00019.54 ATOM 156 O ASP 21 −23.988 −29.627 34.131 1.000 22.49 ATOM 157 NGLY 22 −22.550 −31.250 34.697 1.000 13.19 ATOM 158 CA GLY 22 −23.279−32.377 34.166 1.000 15.71 ATOM 159 C GLY 22 −23.507 −32.377 32.6591.000 20.02 ATOM 160 O GLY 22 −23.370 −33.431 32.036 1.000 23.32 ATOM161 N ALA 23 −23.846 −31.235 32.138 1.000 26.40 ATOM 162 CA ALA 23−24.265 −30.672 30.873 1.000 28.79 ATOM 163 CB ALA 23 −24.483 −29.19231.152 1.000 32.86 ATOM 164 C ALA 23 −23.309 −30.988 29.745 1.000 22.68ATOM 165 O ALA 23 −22.922 −32.189 29.753 1.000 40.02 ATOM 166 N PRO 24−22.847 −30.255 28.748 1.000 12.97 ATOM 167 CD PRO 24 −22.892 −28.85528.309 1.000 15.92 ATOM 168 CA PRO 24 −22.051 −31.028 27.767 1.000 5.31ATOM 169 CB PRO 24 −22.024 −30.134 26.520 1.000 4.03 ATOM 170 CG PRO 24−22.002 −28.762 27.105 1.000 6.80 ATOM 171 C PRO 24 −20.622 −31.27328.222 1.000 14.45 ATOM 172 O PRO 24 −20.034 −30.591 29.056 1.000 19.65ATOM 173 N THR 25 −20.062 −32.310 27.600 1.000 13.21 ATOM 174 CA THR 25−18.685 −32.690 27.894 1.000 11.82 ATOM 175 CB THR 25 −18.691 −33.77228.987 1.000 12.19 ATOM 176 OG1 THR 25 −17.348 −34.104 29.355 1.00019.38 ATOM 177 CG2 THR 25 −19.372 −35.027 28.454 1.000 0.00 ATOM 178 CTHR 25 −18.009 −33.160 26.620 1.000 14.10 ATOM 179 O THR 25 −18.555−33.019 25.518 1.000 16.46 ATOM 180 N GLU 26 −16.818 −33.724 26.7621.000 12.30 ATOM 181 CA GLU 26 −16.157 −34.314 25.598 1.000 13.24 ATOM182 CB GLU 26 −14.909 −33.518 25.225 1.000 15.75 ATOM 183 CG GLU 26−15.211 −32.066 24.873 1.000 25.45 ATOM 184 CD GLU 26 −15.451 −31.15226.056 1.000 27.41 ATOM 185 OE1 GLU 26 −14.687 −31.210 27.048 1.00022.86 ATOM 186 OE2 GLU 26 −16.416 −30.347 26.012 1.000 17.32 ATOM 187 CGLU 26 −15.850 −35.775 25.891 1.000 8.80 ATOM 188 O GLU 26 −16.279−36.316 26.909 1.000 2.55 ATOM 189 N ARG 27 −15.121 −36.421 25.001 1.00013.28 ATOM 190 CA ARG 27 −14.783 −37.838 25.124 1.000 12.71 ATOM 191 CBARG 27 −14.857 −38.447 23.726 1.000 6.07 ATOM 192 CG ARG 27 −14.491−39.908 23.585 1.000 4.38 ATOM 193 CD ARG 27 −14.879 −40.387 22.1861.000 11.29 ATOM 194 NE ARG 27 −14.974 −41.840 22.110 1.000 13.10 ATOM195 CZ ARG 27 −15.191 −42.517 20.992 1.000 9.74 ATOM 196 NH1 ARG 27−15.337 −41.868 19.842 1.000 11.38 ATOM 197 NH2 ARG 27 −15.262 −43.83921.029 1.000 0.00 ATOM 198 C ARG 27 −13.413 −38.031 25.746 1.000 8.79ATOM 199 O ARG 27 −12.534 −37.181 25.579 1.000 17.59 ATOM 200 N PHE 28−13.183 −39.133 26.461 1.000 12.29 ATOM 201 CA PHE 28 −11.826 −39.37926.955 1.000 9.91 ATOM 202 CB PHE 28 −11.783 −40.575 27.900 1.000 10.13ATOM 203 CG PHE 28 −12.084 −40.263 29.355 1.000 11.54 ATOM 204 CD1 PHE28 −11.250 −39.431 30.084 1.000 8.88 ATOM 205 CD2 PHE 28 −13.194 −40.80229.979 1.000 11.27 ATOM 206 CE1 PHE 28 −11.535 −39.156 31.408 1.000 8.90ATOM 207 CE2 PHE 28 −13.486 −40.533 31.305 1.000 5.41 ATOM 208 CZ PHE 28−12.647 −39.703 32.020 1.000 0.61 ATOM 209 C PHE 28 −10.901 −39.63525.770 1.000 11.56 ATOM 210 O PHE 28 −11.370 −40.112 24.736 1.000 13.14ATOM 211 N ALA 29 −9.612 −39.349 25.896 1.000 13.02 ATOM 212 CA ALA 29−8.674 −39.656 24.818 1.000 13.91 ATOM 213 CB ALA 29 −7.275 −39.16325.151 1.000 6.49 ATOM 214 C ALA 29 −8.662 −41.157 24.545 1.000 15.68ATOM 215 O ALA 29 −8.937 −41.954 25.446 1.000 31.74 ATOM 216 N PRO 30−8.345 −41.537 23.314 1.000 11.44 ATOM 217 CD PRO 30 −7.982 −40.66022.192 1.000 12.10 ATOM 218 CA PRO 30 −8.326 −42.955 22.936 1.000 18.85ATOM 219 CB PRO 30 −7.822 −42.956 21.494 1.000 16.38 ATOM 220 CG PRO 30−7.283 −41.593 21.244 1.000 14.74 ATOM 221 C PRO 30 −7.386 −43.76723.826 1.000 13.40 ATOM 222 O PRO 30 −7.570 −44.969 23.979 1.000 8.18ATOM 223 N ASP 31 −6.396 −43.115 24.412 1.000 22.50 ATOM 224 CA ASP 31−5.426 −43.715 25.312 1.000 26.63 ATOM 225 CB ASP 31 −4.170 −42.84125.398 1.000 30.41 ATOM 226 CG ASP 31 −3.792 −42.143 24.108 1.000 39.21ATOM 227 OD1 ASP 31 −2.577 −42.086 23.802 1.000 39.00 ATOM 228 OD2 ASP31 −4.673 −41.634 23.375 1.000 37.50 ATOM 229 C ASP 31 −5.985 −43.92626.721 1.000 17.49 ATOM 230 O ASP 31 −5.482 −44.784 27.450 1.000 25.27ATOM 231 N VAL 32 −6.989 −43.150 27.092 1.000 14.45 ATOM 232 CA VAL 32−7.592 −43.125 28.421 1.000 12.64 ATOM 233 CB VAL 32 −7.966 −41.68328.814 1.000 10.68 ATOM 234 CG1 VAL 32 −8.580 −41.609 30.199 1.000 13.66ATOM 235 CG2 VAL 32 −6.742 −40.774 28.752 1.000 20.51 ATOM 236 C VAL 32−8.808 −44.042 28.507 1.000 9.73 ATOM 237 O VAL 32 −8.890 −44.834 29.4521.000 2.23 ATOM 238 N ARG 33 −9.722 −43.964 27.553 1.000 10.63 ATOM 239CA ARG 33 −10.888 −44.824 27.410 1.000 6.85 ATOM 240 CB ARG 33 −11.369−44.833 25.961 1.000 16.41 ATOM 241 CG ARG 33 −12.281 −43.727 25.4881.000 21.19 ATOM 242 CD ARG 33 −12.464 −43.806 23.974 1.000 26.66 ATOM243 NE ARG 33 −11.862 −42.659 23.309 1.000 30.35 ATOM 244 CZ ARG 33−11.493 −42.567 22.044 1.000 31.60 ATOM 245 NH1 ARG 33 −11.658 −43.58521.214 1.000 34.85 ATOM 246 NH2 ARG 33 −10.952 −41.433 21.610 1.00052.70 ATOM 247 C ARG 33 −10.600 −46.279 27.775 1.000 9.71 ATOM 248 O ARG33 −9.603 −46.830 27.300 1.000 16.85 ATOM 249 N TRP 34 −11.450 −46.92428.577 1.000 10.64 ATOM 250 CA TRP 34 −11.166 −48.311 28.952 1.000 6.46ATOM 251 CB TRP 34 −12.149 −48.855 29.979 1.000 12.45 ATOM 252 CG TRP 34−13.561 −49.106 29.583 1.000 6.95 ATOM 253 CD2 TRP 34 −14.104 −50.19928.835 1.000 9.27 ATOM 254 CE2 TRP 34 −15.493 −49.986 28.723 1.000 5.43ATOM 255 CE3 TRP 34 −13.571 −51.345 28.240 1.000 14.72 ATOM 256 CD1 TRP34 −14.622 −48.298 29.888 1.000 4.49 ATOM 257 NE1 TRP 34 −15.786 −48.82029.374 1.000 4.03 ATOM 258 CZ2 TRP 34 −16.337 −50.864 28.050 1.000 8.19ATOM 259 CZ3 TRP 34 −14.405 −52.216 27.572 1.000 12.73 ATOM 260 CH2 TRP34 −15.778 −51.976 27.479 1.000 8.32 ATOM 261 C TRP 34 −11.111 −49.21427.723 1.000 7.27 ATOM 262 O TRP 34 −10.393 −50.222 27.767 1.000 11.53ATOM 263 N THR 35 −11.839 −48.887 26.659 1.000 1.15 ATOM 264 CA THR 35−11.730 −49.673 25.431 1.000 5.29 ATOM 265 CB THR 35 −12.708 −49.23924.331 1.000 3.10 ATOM 266 OG1 THR 35 −12.629 −47.820 24.163 1.000 15.85ATOM 267 CG2 THR 35 −14.146 −49.549 24.726 1.000 5.16 ATOM 268 C THR 35−10.307 −49.555 24.882 1.000 14.32 ATOM 269 O THR 35 −9.738 −50.49424.333 1.000 12.77 ATOM 270 N GLY 36 −9.756 −48.361 25.060 1.000 15.72ATOM 271 CA GLY 36 −8.392 −48.056 24.689 1.000 15.87 ATOM 272 C GLY 36−7.407 −48.785 25.583 1.000 14.86 ATOM 273 O GLY 36 −6.374 −49.25225.101 1.000 22.97 ATOM 274 N VAL 37 −7.686 −48.905 26.884 1.000 12.48ATOM 275 CA VAL 37 −6.696 −49.577 27.728 1.000 11.76 ATOM 276 CB VAL 37−6.921 −49.365 29.229 1.000 10.95 ATOM 277 CG1 VAL 37 −6.092 −50.38230.009 1.000 0.00 ATOM 278 CG2 VAL 37 −6.577 −47.940 29.630 1.000 10.31ATOM 279 C VAL 37 −6.707 −51.081 27.471 1.000 16.75 ATOM 280 O VAL 37−5.669 −51.735 27.494 1.000 14.29 ATOM 281 N LEU 38 −7.911 −51.58627.238 1.000 14.60 ATOM 282 CA LEU 38 −8.094 −52.999 26.917 1.000 11.25ATOM 283 CB LEU 38 −9.573 −53.266 26.660 1.000 12.92 ATOM 284 CG LEU 38−9.975 −54.663 26.198 1.000 15.77 ATOM 285 CD1 LEU 38 −9.747 −55.69127.293 1.000 0.00 ATOM 286 CD2 LEU 38 −11.425 −54.677 25.733 1.000 24.28ATOM 287 C LEU 38 −7.224 −53.347 25.720 1.000 7.67 ATOM 288 O LEU 38−6.408 −54.262 25.740 1.000 13.04 ATOM 289 N ALA 39 −7.404 −52.56824.659 1.000 9.64 ATOM 290 CA ALA 39 −6.603 −52.667 23.451 1.000 3.53ATOM 291 CB ALA 39 −6.894 −51.487 22.530 1.000 6.32 ATOM 292 C ALA 39−5.112 −52.704 23.761 1.000 9.32 ATOM 293 O ALA 39 −4.411 −53.632 23.3671.000 28.59 ATOM 294 N GLN 40 −4.653 −51.665 24.456 1.000 21.51 ATOM 295CA GLN 40 −3.251 −51.553 24.833 1.000 18.93 ATOM 296 CB GLN 40 −2.974−50.365 25.744 1.000 28.00 ATOM 297 CG GLN 40 −3.597 −49.034 25.3781.000 37.51 ATOM 298 CD GLN 40 −3.070 −47.877 26.214 1.000 40.85 ATOM299 OE1 GLN 40 −1.998 −47.335 25.933 1.000 61.34 ATOM 300 NE2 GLN 40−3.809 −47.475 27.248 1.000 9.83 ATOM 301 C GLN 40 −2.822 −52.851 25.5251.000 10.96 ATOM 302 O GLN 40 −1.856 −53.475 25.106 1.000 18.66 ATOM 303N GLN 41 −3.563 −53.239 26.552 1.000 15.02 ATOM 304 CA GLN 41 −3.253−54.423 27.337 1.000 22.27 ATOM 305 CB GLN 41 −4.258 −54.582 28.4841.000 16.69 ATOM 306 CG GLN 41 −4.064 −53.605 29.624 1.000 14.55 ATOM307 CD GLN 41 −2.788 −53.852 30.406 1.000 16.86 ATOM 308 OE1 GLN 41−2.759 −54.650 31.344 1.000 13.75 ATOM 309 NE2 GLN 41 −1.731 −53.15830.008 1.000 21.79 ATOM 310 C GLN 41 −3.261 −55.694 26.493 1.000 28.40ATOM 311 O GLN 41 −2.442 −56.589 26.703 1.000 26.71 ATOM 312 N LEU 42−4.190 −55.776 25.546 1.000 28.62 ATOM 313 CA LEU 42 −4.373 −57.00724.780 1.000 26.50 ATOM 314 CB LEU 42 −5.707 −56.920 24.012 1.000 19.31ATOM 315 CG LEU 42 −6.934 −57.122 24.914 1.000 16.32 ATOM 316 CD1 LEU 42−8.226 −57.077 24.119 1.000 10.94 ATOM 317 CD2 LEU 42 −6.810 −58.43825.673 1.000 15.03 ATOM 318 C LEU 42 −3.217 −57.312 23.846 1.000 23.29ATOM 319 O LEU 42 −2.770 −58.457 23.728 1.000 20.82 ATOM 320 N GLY 43−2.693 −56.312 23.141 1.000 22.18 ATOM 321 CA GLY 43 −1.605 −56.59022.215 1.000 18.95 ATOM 322 C GLY 43 −2.086 −56.793 20.791 1.000 23.97ATOM 323 O GLY 43 −3.284 −56.838 20.514 1.000 27.50 ATOM 324 N ALA 44−1.136 −56.927 19.879 1.000 22.72 ATOM 325 CA ALA 44 −1.317 −57.01218.448 1.000 24.25 ATOM 326 CB ALA 44 0.048 −56.939 17.755 1.000 13.44ATOM 327 C ALA 44 −2.034 −58.272 17.990 1.000 23.83 ATOM 328 O ALA 44−2.146 −58.520 16.787 1.000 17.77 ATOM 329 N ASP 45 −2.524 −59.08618.917 1.000 21.59 ATOM 330 CA ASP 45 −3.230 −60.298 18.495 1.000 17.80ATOM 331 CB ASP 45 −2.705 −61.491 19.296 1.000 18.22 ATOM 332 CG ASP 45−1.201 −61.625 19.113 1.000 24.69 ATOM 333 OD1 ASP 45 −0.710 −61.17418.053 1.000 34.10 ATOM 334 OD2 ASP 45 −0.517 −62.159 20.007 1.000 33.14ATOM 335 C ASP 45 −4.732 −60.107 18.647 1.000 11.82 ATOM 336 O ASP 45−5.535 −60.992 18.364 1.000 23.89 ATOM 337 N PHE 46 −5.097 −58.91419.097 1.000 9.27 ATOM 338 CA PHE 46 −6.485 −58.519 19.253 1.000 12.25ATOM 339 CB PHE 46 −6.909 −58.479 20.722 1.000 14.52 ATOM 340 CG PHE 46−6.474 −59.693 21.529 1.000 11.99 ATOM 341 CD1 PHE 46 −5.160 −59.81421.956 1.000 12.17 ATOM 342 CD2 PHE 46 −7.383 −60.690 21.846 1.000 8.34ATOM 343 CE1 PHE 46 −4.760 −60.917 22.683 1.000 13.46 ATOM 344 CE2 PHE46 −6.990 −61.794 22.575 1.000 6.30 ATOM 345 CZ PHE 46 −5.680 −61.90422.998 1.000 8.44 ATOM 346 C PHE 46 −6.725 −57.149 18.615 1.000 13.30ATOM 347 O PHE 46 −5.816 −56.366 18.366 1.000 27.22 ATOM 348 N GLU 47−7.992 −56.883 18.349 1.000 12.78 ATOM 349 CA GLU 47 −8.469 −55.61617.833 1.000 9.15 ATOM 350 CB GLU 47 −8.667 −55.644 16.325 1.000 11.20ATOM 351 CG GLU 47 −8.791 −54.276 15.670 1.000 21.84 ATOM 352 CD GLU 47−9.726 −54.293 14.474 1.000 25.88 ATOM 353 OE1 GLU 47 −9.575 −55.20513.632 1.000 30.74 ATOM 354 OE2 GLU 47 −10.602 −53.408 14.388 1.000 7.59ATOM 355 C GLU 47 −9.781 −55.280 18.550 1.000 11.37 ATOM 356 O GLU 47−10.722 −56.071 18.545 1.000 11.73 ATOM 357 N VAL 48 −9.775 −54.10319.160 1.000 10.53 ATOM 358 CA VAL 48 −10.954 −53.604 19.843 1.000 8.11ATOM 359 CB VAL 48 −10.595 −52.826 21.115 1.000 9.71 ATOM 360 CG1 VAL 48−11.842 −52.251 21.773 1.000 15.31 ATOM 361 CG2 VAL 48 −9.849 −53.73222.085 1.000 7.41 ATOM 362 C VAL 48 −11.745 −52.714 18.882 1.000 12.72ATOM 363 O VAL 48 −11.147 −51.879 18.203 1.000 10.16 ATOM 364 N ILE 49−13.046 −52.943 18.862 1.000 13.04 ATOM 365 CA ILE 49 −14.031 −52.17018.122 1.000 14.10 ATOM 366 CB ILE 49 −14.879 −53.068 17.203 1.000 16.77ATOM 367 CG2 ILE 49 −15.735 −52.214 16.285 1.000 1.57 ATOM 368 CG1 ILE49 −14.049 −54.081 16.415 1.000 18.10 ATOM 369 CD1 ILE 49 −14.687−54.559 15.133 1.000 14.33 ATOM 370 C ILE 49 −14.930 −51.406 19.0911.000 9.02 ATOM 371 O ILE 49 −15.531 −52.013 19.983 1.000 15.82 ATOM 372N GLU 50 −15.000 −50.085 18.932 1.000 5.34 ATOM 373 CA GLU 50 −15.730−49.277 19.911 1.000 12.03 ATOM 374 CB GLU 50 −14.967 −47.984 20.2221.000 10.36 ATOM 375 CG GLU 50 −13.623 −48.203 20.889 1.000 7.32 ATOM376 CD GLU 50 −12.768 −46.966 21.056 1.000 7.06 ATOM 377 OE1 GLU 50−12.744 −46.077 20.177 1.000 5.78 ATOM 378 OE2 GLU 50 −12.079 −46.87022.101 1.000 25.19 ATOM 379 C GLU 50 −17.145 −48.962 19.446 1.000 6.79ATOM 380 O GLU 50 −17.358 −48.318 18.423 1.000 8.80 ATOM 381 N GLU 51−18.118 −49.429 20.225 1.000 9.34 ATOM 382 CA GLU 51 −19.524 −49.17919.924 1.000 16.23 ATOM 383 CB GLU 51 −20.173 −50.400 19.270 1.000 15.22ATOM 384 CG GLU 51 −19.757 −50.596 17.820 1.000 18.39 ATOM 385 CD GLU 51−20.348 −49.531 16.917 1.000 17.99 ATOM 386 OE1 GLU 51 −21.352 −48.91217.332 1.000 26.29 ATOM 387 OE2 GLU 51 −19.820 −49.309 15.809 1.00015.93 ATOM 388 C GLU 51 −20.295 −48.788 21.184 1.000 10.51 ATOM 389 OGLU 51 −21.202 −49.495 21.623 1.000 7.29 ATOM 390 N GLY 52 −19.906−47.655 21.751 1.000 5.90 ATOM 391 CA GLY 52 −20.533 −47.140 22.9611.000 3.93 ATOM 392 C GLY 52 −21.329 −45.887 22.635 1.000 6.21 ATOM 393O GLY 52 −20.785 −44.950 22.057 1.000 16.40 ATOM 394 N LEU 53 −22.607−45.890 22.989 1.000 11.68 ATOM 395 CA LEU 53 −23.498 −44.764 22.7101.000 7.60 ATOM 396 CB LEU 53 −24.627 −45.195 21.792 1.000 4.45 ATOM 397CG LEU 53 −25.576 −44.164 21.185 1.000 3.84 ATOM 398 CD1 LEU 53 −26.721−43.872 22.141 1.000 15.09 ATOM 399 CD2 LEU 53 −24.856 −42.874 20.8171.000 3.41 ATOM 400 C LEU 53 −24.035 −44.204 24.023 1.000 5.05 ATOM 401O LEU 53 −24.664 −44.920 24.801 1.000 5.74 ATOM 402 N SER 54 −23.771−42.918 24.251 1.000 9.85 ATOM 403 CA SER 54 −24.192 −42.296 25.5021.000 10.24 ATOM 404 CB SER 54 −23.797 −40.819 25.524 1.000 7.63 ATOM405 OG SER 54 −22.395 −40.683 25.640 1.000 4.65 ATOM 406 C SER 54−25.695 −42.448 25.691 1.000 7.74 ATOM 407 O SER 54 −26.438 −42.32624.717 1.000 10.39 ATOM 408 N ALA 55 −26.127 −42.713 26.920 1.000 0.00ATOM 409 CA ALA 55 −27.554 −42.749 27.218 1.000 0.00 ATOM 410 CB ALA 55−28.209 −41.474 26.713 1.000 0.00 ATOM 411 C ALA 55 −28.235 −43.98226.640 1.000 6.11 ATOM 412 O ALA 55 −29.442 −44.179 26.816 1.000 2.57ATOM 413 N ARG 56 −27.474 −44.843 25.971 1.000 8.50 ATOM 414 CA ARG 56−27.997 −46.084 25.433 1.000 5.94 ATOM 415 CB ARG 56 −26.919 −46.86824.672 1.000 0.00 ATOM 416 CG ARG 56 −27.420 −48.244 24.247 1.000 2.73ATOM 417 CD ARG 56 −26.467 −48.951 23.307 1.000 0.00 ATOM 418 NE ARG 56−26.552 −48.440 21.935 1.000 6.44 ATOM 419 CZ ARG 56 −25.465 −48.32521.170 1.000 11.18 ATOM 420 NH1 ARG 56 −24.283 −48.678 21.666 1.000 0.00ATOM 421 NH2 ARG 56 −25.549 −47.861 19.928 1.000 1.13 ATOM 422 C ARG 56−28.539 −47.009 26.526 1.000 12.43 ATOM 423 O ARG 56 −27.886 −47.17927.556 1.000 10.16 ATOM 424 N THR 57 −29.697 −47.592 26.262 1.000 9.24ATOM 425 CA THR 57 −30.376 −48.548 27.120 1.000 9.36 ATOM 426 CB THR 57−31.855 −48.161 27.315 1.000 4.78 ATOM 427 OG1 THR 57 −32.608 −48.50926.146 1.000 3.70 ATOM 428 CG2 THR 57 −31.992 −46.656 27.484 1.000 0.00ATOM 429 C THR 57 −30.284 −49.953 26.532 1.000 10.18 ATOM 430 O THR 57−29.873 −50.099 25.378 1.000 12.60 ATOM 431 N THR 58 −30.648 −50.98727.286 1.000 5.87 ATOM 432 CA THR 58 −30.574 −52.349 26.769 1.000 1.65ATOM 433 CB THR 58 −30.850 −53.410 27.853 1.000 5.35 ATOM 434 OG1 THR 58−32.151 −53.196 28.413 1.000 12.48 ATOM 435 CG2 THR 58 −29.859 −53.31129.002 1.000 11.47 ATOM 436 C THR 58 −31.556 −52.569 25.624 1.000 1.31ATOM 437 O THR 58 −31.162 −52.902 24.506 1.000 7.78 ATOM 438 N ASN 59−32.856 −52.404 25.867 1.000 4.91 ATOM 439 CA ASN 59 −33.810 −52.60424.772 1.000 11.25 ATOM 440 CB ASN 59 −34.150 −54.090 24.624 1.000 9.19ATOM 441 CG ASN 59 −35.186 −54.548 25.629 1.000 9.50 ATOM 442 OD1 ASN 59−35.293 −54.000 26.725 1.000 13.36 ATOM 443 ND2 ASN 59 −35.965 −55.55625.263 1.000 4.31 ATOM 444 C ASN 59 −35.070 −51.775 24.960 1.000 8.67ATOM 445 O ASN 59 −36.172 −52.160 24.574 1.000 12.75 ATOM 446 N ILE 60−34.938 −50.587 25.548 1.000 10.46 ATOM 447 CA ILE 60 −36.128 −49.75225.722 1.000 10.70 ATOM 448 CB ILE 60 −36.572 −49.721 27.198 1.000 11.36ATOM 449 CG2 ILE 60 −35.465 −49.223 28.112 1.000 0.00 ATOM 450 CG1 ILE60 −37.872 −48.940 27.417 1.000 8.05 ATOM 451 CD1 ILE 60 −38.291 −48.80028.860 1.000 27.90 ATOM 452 C ILE 60 −35.879 −48.350 25.177 1.000 16.37ATOM 453 O ILE 60 −34.813 −47.773 25.374 1.000 28.53 ATOM 454 N ASP 61−36.861 −47.811 24.470 1.000 18.37 ATOM 455 CA ASP 61 −36.838 −46.52023.821 1.000 12.62 ATOM 456 CB ASP 61 −38.110 −46.353 22.977 1.000 12.58ATOM 457 CG ASP 61 −38.111 −47.199 21.725 1.000 12.09 ATOM 458 OD1 ASP61 −37.044 −47.723 21.349 1.000 16.37 ATOM 459 OD2 ASP 61 −39.197−47.332 21.122 1.000 23.20 ATOM 460 C ASP 61 −36.796 −45.350 24.7941.000 11.54 ATOM 461 O ASP 61 −37.626 −45.279 25.702 1.000 8.66 ATOM 462N ASP 62 −35.860 −44.428 24.603 1.000 8.03 ATOM 463 CA ASP 62 −35.844−43.228 25.431 1.000 14.39 ATOM 464 CB ASP 62 −34.430 −42.656 25.5651.000 13.94 ATOM 465 CG ASP 62 −34.384 −41.598 26.656 1.000 18.06 ATOM466 OD1 ASP 62 −33.609 −41.768 27.622 1.000 13.05 ATOM 467 OD2 ASP 62−35.129 −40.604 26.536 1.000 20.19 ATOM 468 C ASP 62 −36.759 −42.16224.844 1.000 13.14 ATOM 469 O ASP 62 −36.506 −41.698 23.731 1.000 14.36ATOM 470 N PRO 63 −37.800 −41.751 25.553 1.000 8.49 ATOM 471 CD PRO 63−38.102 −42.088 26.951 1.000 4.73 ATOM 472 CA PRO 63 −38.805 −40.85324.972 1.000 16.60 ATOM 473 CB PRO 63 −39.802 −40.646 26.123 1.000 11.61ATOM 474 CG PRO 63 −39.020 −40.960 27.352 1.000 8.04 ATOM 475 C PRO 63−38.251 −39.504 24.531 1.000 19.70 ATOM 476 O PRO 63 −38.924 −38.73823.835 1.000 10.26 ATOM 477 N THR 64 −37.024 −39.180 24.922 1.000 22.29ATOM 478 CA THR 64 −36.429 −37.908 24.534 1.000 19.30 ATOM 479 CB THR 64−35.852 −37.191 25.769 1.000 20.62 ATOM 480 OG1 THR 64 −34.550 −37.71326.045 1.000 30.42 ATOM 481 CG2 THR 64 −36.718 −37.467 26.992 1.000 7.89ATOM 482 C THR 64 −35.329 −38.087 23.497 1.000 19.22 ATOM 483 O THR 64−34.609 −37.132 23.183 1.000 11.15 ATOM 484 N ASP 65 −35.189 −39.30122.965 1.000 15.61 ATOM 485 CA ASP 65 −34.139 −39.542 21.967 1.000 18.78ATOM 486 CB ASP 65 −32.777 −39.286 22.605 1.000 20.50 ATOM 487 CG ASP 65−31.613 −39.348 21.638 1.000 17.33 ATOM 488 OD1 ASP 65 −31.767 −39.93520.550 1.000 19.33 ATOM 489 OD2 ASP 65 −30.538 −38.810 21.983 1.00015.26 ATOM 490 C ASP 65 −34.241 −40.945 21.382 1.000 14.84 ATOM 491 OASP 65 −33.982 −41.936 22.060 1.000 8.38 ATOM 492 N PRO 66 −34.638−41.026 20.115 1.000 15.75 ATOM 493 CD PRO 66 −34.896 −39.870 19.2351.000 23.61 ATOM 494 CA PRO 66 −34.882 −42.301 19.441 1.000 9.14 ATOM495 CB PRO 66 −35.693 −41.871 18.206 1.000 14.38 ATOM 496 CG PRO 66−35.210 −40.494 17.902 1.000 16.45 ATOM 497 C PRO 66 −33.621 −43.02918.995 1.000 8.15 ATOM 498 O PRO 66 −33.695 −44.041 18.283 1.000 12.38ATOM 499 N ARG 67 −32.446 −42.557 19.404 1.000 11.98 ATOM 500 CA ARG 67−31.209 −43.225 19.020 1.000 7.77 ATOM 501 CB ARG 67 −30.081 −42.21118.831 1.000 8.16 ATOM 502 CG ARG 67 −30.162 −41.308 17.614 1.000 7.27ATOM 503 CD ARG 67 −29.078 −40.228 17.713 1.000 11.05 ATOM 504 NE ARG 67−29.378 −39.266 18.769 1.000 11.17 ATOM 505 CZ ARG 67 −28.768 −38.11519.001 1.000 13.35 ATOM 506 NH1 ARG 67 −27.756 −37.708 18.245 1.000 3.80ATOM 507 NH2 ARG 67 −29.168 −37.347 20.010 1.000 9.93 ATOM 508 C ARG 67−30.728 −44.239 20.048 1.000 8.92 ATOM 509 O ARG 67 −29.714 −44.88719.774 1.000 13.65 ATOM 510 N LEU 68 −31.389 −44.365 21.191 1.000 9.14ATOM 511 CA LEU 68 −30.805 −45.057 22.335 1.000 13.92 ATOM 512 CB LEU 68−31.052 −44.223 23.608 1.000 7.80 ATOM 513 CG LEU 68 −30.899 −42.70723.481 1.000 8.78 ATOM 514 CD1 LEU 68 −31.285 −41.987 24.770 1.000 13.12ATOM 515 CD2 LEU 68 −29.477 −42.333 23.090 1.000 3.77 ATOM 516 C LEU 68−31.299 −46.478 22.571 1.000 16.19 ATOM 517 O LEU 68 −30.895 −47.09223.574 1.000 5.21 ATOM 518 N ASN 69 −32.139 −47.056 21.716 1.000 7.75ATOM 519 CA ASN 69 −32.520 −48.457 21.927 1.000 6.53 ATOM 520 CB ASN 69−33.807 −48.842 21.198 1.000 6.25 ATOM 521 CG ASN 69 −34.377 −50.17221.658 1.000 11.70 ATOM 522 OD1 ASN 69 −33.732 −51.219 21.664 1.000 2.64ATOM 523 ND2 ASN 69 −35.646 −50.164 22.057 1.000 10.84 ATOM 524 C ASN 69−31.406 −49.404 21.480 1.000 8.62 ATOM 525 O ASN 69 −31.204 −49.61720.287 1.000 14.61 ATOM 526 N GLY 70 −30.697 −49.972 22.452 1.000 8.79ATOM 527 CA GLY 70 −29.582 −50.854 22.212 1.000 1.64 ATOM 528 C GLY 70−29.911 −52.031 21.316 1.000 6.17 ATOM 529 O GLY 70 −29.189 −52.29320.355 1.000 12.06 ATOM 530 N ALA 71 −30.982 −52.744 21.622 1.000 1.39ATOM 531 CA ALA 71 −31.442 −53.885 20.843 1.000 5.92 ATOM 532 CB ALA 71−32.688 −54.457 21.529 1.000 3.81 ATOM 533 C ALA 71 −31.766 −53.56519.392 1.000 4.67 ATOM 534 O ALA 71 −31.565 −54.391 18.490 1.000 0.00ATOM 535 N SER 72 −32.295 −52.371 19.121 1.000 3.88 ATOM 536 CA SER 72−32.687 −52.033 17.752 1.000 6.33 ATOM 537 CB SER 72 −33.678 −50.87017.759 1.000 4.05 ATOM 538 OG SER 72 −33.023 −49.637 18.004 1.000 25.62ATOM 539 C SER 72 −31.468 −51.730 16.884 1.000 7.90 ATOM 540 O SER 72−31.568 −51.720 15.658 1.000 12.06 ATOM 541 N TYR 73 −30.315 −51.50517.498 1.000 8.51 ATOM 542 CA TYR 73 −29.070 −51.210 16.789 1.000 8.77ATOM 543 CB TYR 73 −28.394 −50.029 17.478 1.000 10.31 ATOM 544 CG TYR 73−27.124 −49.453 16.913 1.000 11.92 ATOM 545 CD1 TYR 73 −27.113 −48.32916.090 1.000 8.49 ATOM 546 CE1 TYR 73 −25.931 −47.812 15.586 1.000 1.47ATOM 547 CD2 TYR 73 −25.888 −50.018 17.201 1.000 10.36 ATOM 548 CE2 TYR73 −24.704 −49.512 16.703 1.000 9.07 ATOM 549 CZ TYR 73 −24.727 −48.39815.890 1.000 5.36 ATOM 550 OH TYR 73 −23.544 −47.902 15.391 1.000 10.80ATOM 551 C TYR 73 −28.148 −52.419 16.730 1.000 13.31 ATOM 552 O TYR 73−27.404 −52.630 15.764 1.000 10.40 ATOM 553 N LEU 74 −28.172 −53.26117.759 1.000 8.99 ATOM 554 CA LEU 74 −27.204 −54.342 17.901 1.000 7.76ATOM 555 CB LEU 74 −27.554 −55.155 19.155 1.000 9.47 ATOM 556 CG LEU 74−26.402 −55.532 20.080 1.000 10.36 ATOM 557 CD1 LEU 74 −26.786 −56.72920.939 1.000 25.33 ATOM 558 CD2 LEU 74 −25.137 −55.819 19.288 1.00013.92 ATOM 559 C LEU 74 −27.088 −55.253 16.687 1.000 5.72 ATOM 560 O LEU74 −25.980 −55.383 16.141 1.000 7.01 ATOM 561 N PRO 75 −28.141 −55.90716.219 1.000 6.99 ATOM 562 CD PRO 75 −29.553 −55.794 16.615 1.000 1.55ATOM 563 CA PRO 75 −27.965 −56.896 15.140 1.000 7.57 ATOM 564 CB PRO 75−29.384 −57.401 14.855 1.000 5.01 ATOM 565 CG PRO 75 −30.158 −57.06316.086 1.000 6.27 ATOM 566 C PRO 75 −27.364 −56.285 13.882 1.000 4.16ATOM 567 O PRO 75 −26.651 −56.971 13.158 1.000 4.35 ATOM 568 N SER 76−27.640 −55.014 13.615 1.000 6.22 ATOM 569 CA SER 76 −27.050 −54.32212.473 1.000 0.00 ATOM 570 CB SER 76 −27.758 −52.978 12.261 1.000 0.00ATOM 571 OG SER 76 −29.120 −53.249 11.920 1.000 0.00 ATOM 572 C SER 76−25.554 −54.127 12.674 1.000 0.69 ATOM 573 O SER 76 −24.767 −54.28011.740 1.000 4.06 ATOM 574 N CYS 77 −25.202 −53.802 13.911 1.000 2.82ATOM 575 CA CYS 77 −23.851 −53.599 14.384 1.000 2.99 ATOM 576 CB CYS 77−23.878 −53.202 15.868 1.000 0.00 ATOM 577 SG CYS 77 −22.325 −52.50816.451 1.000 8.78 ATOM 578 C CYS 77 −22.962 −54.831 14.225 1.000 13.77ATOM 579 O CYS 77 −21.828 −54.700 13.755 1.000 12.12 ATOM 580 N LEU 78−23.455 −55.996 14.621 1.000 15.71 ATOM 581 CA LEU 78 −22.751 −57.26814.538 1.000 10.13 ATOM 582 CB LEU 78 −23.617 −58.387 15.129 1.000 2.73ATOM 583 CG LEU 78 −23.777 −58.354 16.651 1.000 7.98 ATOM 584 CD1 LEU 78−24.866 −59.319 17.085 1.000 3.36 ATOM 585 CD2 LEU 78 −22.451 −58.67617.330 1.000 8.53 ATOM 586 C LEU 78 −22.385 −57.650 13.106 1.000 9.88ATOM 587 O LEU 78 −21.222 −57.855 12.761 1.000 12.55 ATOM 588 N ALA 79−23.407 −57.748 12.271 1.000 11.93 ATOM 589 CA ALA 79 −23.297 −58.02210.848 1.000 2.98 ATOM 590 CB ALA 79 −24.699 −58.042 10.255 1.000 0.32ATOM 591 C ALA 79 −22.393 −57.026 10.127 1.000 7.73 ATOM 592 O ALA 79−21.724 −57.408 9.163 1.000 13.15 ATOM 593 N THR 80 −22.337 −55.77410.560 1.000 10.93 ATOM 594 CA THR 80 −21.427 −54.757 10.044 1.000 6.56ATOM 595 CB THR 80 −21.703 −53.373 10.669 1.000 9.10 ATOM 596 OG1 THR 80−23.013 −52.897 10.320 1.000 4.47 ATOM 597 CG2 THR 80 −20.722 −52.32810.148 1.000 8.02 ATOM 598 C THR 80 −19.970 −55.117 10.317 1.000 10.87ATOM 599 O THR 80 −19.103 −55.052 9.450 1.000 12.66 ATOM 600 N HIS 81−19.659 −55.512 11.548 1.000 13.90 ATOM 601 CA HIS 81 −18.282 −55.72011.978 1.000 13.04 ATOM 602 CB HIS 81 −18.119 −55.195 13.418 1.000 15.15ATOM 603 CG HIS 81 −18.279 −53.704 13.502 1.000 10.10 ATOM 604 CD2 HIS81 −19.202 −52.927 14.111 1.000 6.25 ATOM 605 ND1 HIS 81 −17.404 −52.83312.889 1.000 7.20 ATOM 606 CE1 HIS 81 −17.775 −51.589 13.117 1.000 7.73ATOM 607 NE2 HIS 81 −18.867 −51.616 13.863 1.000 6.24 ATOM 608 C HIS 81−17.827 −57.166 11.896 1.000 9.61 ATOM 609 O HIS 81 −16.674 −57.46012.216 1.000 10.35 ATOM 610 N LEU 82 −18.689 −58.081 11.470 1.000 4.74ATOM 611 CA LEU 82 −18.257 −59.461 11.247 1.000 6.06 ATOM 612 CB LEU 82−19.399 −60.263 10.631 1.000 6.90 ATOM 613 CG LEU 82 −20.535 −60.71611.541 1.000 6.83 ATOM 614 CD1 LEU 82 −21.388 −61.774 10.851 1.000 11.79ATOM 615 CD2 LEU 82 −19.987 −61.246 12.856 1.000 23.45 ATOM 616 C LEU 82−17.042 −59.500 10.337 1.000 6.51 ATOM 617 O LEU 82 −16.972 −58.7229.375 1.000 1.45 ATOM 618 N PRO 83 −16.056 −60.360 10.556 1.000 7.15ATOM 619 CD PRO 83 −14.823 −60.374 9.731 1.000 0.00 ATOM 620 CA PRO 83−16.043 −61.394 11.583 1.000 5.44 ATOM 621 CB PRO 83 −14.941 −62.34111.067 1.000 9.33 ATOM 622 CG PRO 83 −13.968 −61.405 10.415 1.000 7.09ATOM 623 C PRO 83 −15.638 −60.922 12.973 1.000 10.31 ATOM 624 O PRO 83−14.716 −60.125 13.110 1.000 16.21 ATOM 625 N LEU 84 −16.319 −61.43413.994 1.000 14.34 ATOM 626 CA LEU 84 −16.009 −61.132 15.382 1.000 10.66ATOM 627 CB LEU 84 −17.165 −60.373 16.049 1.000 7.23 ATOM 628 CG LEU 84−17.485 −59.010 15.434 1.000 2.01 ATOM 629 CD1 LEU 84 −18.843 −58.51815.902 1.000 8.19 ATOM 630 CD2 LEU 84 −16.382 −58.019 15.766 1.000 5.93ATOM 631 C LEU 84 −15.734 −62.386 16.203 1.000 7.34 ATOM 632 O LEU 84−16.299 −63.447 15.945 1.000 8.40 ATOM 633 N ASP 85 −14.879 −62.24717.208 1.000 8.68 ATOM 634 CA ASP 85 −14.607 −63.332 18.146 1.000 10.21ATOM 635 CB ASP 85 −13.093 −63.433 18.382 1.000 15.96 ATOM 636 CG ASP 85−12.338 −63.789 17.117 1.000 11.01 ATOM 637 OD1 ASP 85 −12.343 −64.97516.727 1.000 9.49 ATOM 638 OD2 ASP 85 −11.739 −62.878 16.518 1.000 28.18ATOM 639 C ASP 85 −15.313 −63.142 19.477 1.000 0.00 ATOM 640 O ASP 85−15.778 −64.067 20.137 1.000 5.48 ATOM 641 N LEU 86 −15.414 −61.90719.958 1.000 7.62 ATOM 642 CA LEU 86 −16.080 −61.695 21.243 1.000 8.84ATOM 643 CB LEU 86 −15.085 −61.690 22.403 1.000 12.15 ATOM 644 CG LEU 86−15.655 −61.580 23.822 1.000 13.98 ATOM 645 CD1 LEU 86 −16.562 −62.75724.151 1.000 7.12 ATOM 646 CD2 LEU 86 −14.535 −61.477 24.850 1.000 10.28ATOM 647 C LEU 86 −16.841 −60.374 21.221 1.000 6.69 ATOM 648 O LEU 86−16.327 −59.409 20.649 1.000 8.05 ATOM 649 N VAL 87 −18.013 −60.36121.842 1.000 4.26 ATOM 650 CA VAL 87 −18.752 −59.127 22.049 1.000 2.21ATOM 651 CB VAL 87 −20.150 −59.126 21.413 1.000 8.44 ATOM 652 CG1 VAL 87−20.848 −57.808 21.722 1.000 2.51 ATOM 653 CG2 VAL 87 −20.104 −59.35219.911 1.000 0.00 ATOM 654 C VAL 87 −18.893 −58.869 23.551 1.000 7.05ATOM 655 O VAL 87 −19.472 −59.660 24.289 1.000 5.76 ATOM 656 N ILE 88−18.351 −57.746 24.010 1.000 7.24 ATOM 657 CA ILE 88 −18.499 −57.33625.400 1.000 6.18 ATOM 658 CB ILE 88 −17.233 −56.652 25.938 1.000 6.54ATOM 659 CG2 ILE 88 −17.458 −56.098 27.333 1.000 11.40 ATOM 660 CG1 ILE88 −16.001 −57.559 25.902 1.000 6.21 ATOM 661 CD1 ILE 88 −14.734 −56.85626.339 1.000 7.20 ATOM 662 C ILE 88 −19.693 −56.394 25.506 1.000 4.68ATOM 663 O ILE 88 −19.817 −55.458 24.716 1.000 10.14 ATOM 664 N ILE 89−20.574 −56.672 26.457 1.000 7.74 ATOM 665 CA ILE 89 −21.765 −55.85726.645 1.000 12.20 ATOM 666 CB ILE 89 −23.052 −56.635 26.306 1.000 12.51ATOM 667 CG2 ILE 89 −24.253 −55.703 26.339 1.000 11.52 ATOM 668 CG1 ILE89 −22.981 −57.390 24.979 1.000 6.47 ATOM 669 CD1 ILE 89 −24.250 −58.11124.597 1.000 8.71 ATOM 670 C ILE 89 −21.861 −55.340 28.078 1.000 11.05ATOM 671 O ILE 89 −22.169 −56.106 28.989 1.000 3.02 ATOM 672 N MET 90−21.590 −54.049 28.236 1.000 7.01 ATOM 673 CA MET 90 −21.808 −53.35929.492 1.000 11.48 ATOM 674 CB MET 90 −20.535 −52.721 30.043 1.000 9.27ATOM 675 CG MET 90 −20.756 −52.097 31.415 1.000 10.33 ATOM 676 XD MET 90−19.202 −51.706 32.246 1.000 17.92 ATOM 677 CE MET 90 −18.544 −50.47531.124 1.000 12.70 ATOM 678 C MET 90 −22.872 −52.262 29.325 1.000 12.90ATOM 679 O MET 90 −22.524 −51.143 28.954 1.000 0.00 ATOM 680 N LEU 91−24.108 −52.639 29.604 1.000 8.70 ATOM 681 CA LEU 91 −25.292 −51.80229.511 1.000 10.58 ATOM 682 CB LEU 91 −26.114 −52.105 28.254 1.000 9.42ATOM 683 CG LEU 91 −25.573 −51.564 26.932 1.000 4.10 ATOM 684 CD1 LEU 91−26.427 −52.046 25.772 1.000 0.00 ATOM 685 CD2 LEU 91 −25.506 −50.04426.961 1.000 2.02 ATOM 686 C LEU 91 −26.169 −52.031 30.734 1.000 2.21ATOM 687 O LEU 91 −25.989 −53.066 31.388 1.000 10.59 ATOM 688 N GLY 92−27.087 −51.117 31.025 1.000 4.69 ATOM 689 CA GLY 92 −27.963 −51.32132.172 1.000 7.16 ATOM 690 C GLY 92 −28.189 −50.092 33.027 1.000 0.00ATOM 691 O GLY 92 −29.266 −49.924 33.603 1.000 8.09 ATOM 692 N THR 93−27.204 −49.219 33.133 1.000 0.16 ATOM 693 CA THR 93 −27.241 −48.00533.929 1.000 9.42 ATOM 694 CB THR 93 −25.927 −47.205 33.768 1.000 17.05ATOM 695 OG1 THR 93 −24.811 −48.063 34.024 1.000 26.81 ATOM 696 CG2 THR93 −25.847 −46.068 34.778 1.000 0.34 ATOM 697 C THR 93 −28.386 −47.07533.551 1.000 9.26 ATOM 698 O THR 93 −29.037 −46.491 34.419 1.000 14.18ATOM 699 N ASN 94 −28.614 −46.927 32.250 1.000 0.69 ATOM 700 CA ASN 94−29.609 −45.981 31.755 1.000 5.12 ATOM 701 CB ASN 94 −29.333 −45.67730.274 1.000 9.42 ATOM 702 CG ASN 94 −27.990 −44.983 30.120 1.000 10.74ATOM 703 OD1 ASN 94 −27.679 −44.062 30.873 1.000 21.66 ATOM 704 ND2 ASN94 −27.175 −45.417 29.174 1.000 18.23 ATOM 705 C ASN 94 −31.029 −46.48131.986 1.000 5.80 ATOM 706 O ASN 94 −31.889 −45.654 32.317 1.000 4.04ATOM 707 N ASP 95 −31.282 −47.777 31.863 1.000 4.02 ATOM 708 CA ASP 95−32.568 −48.411 32.137 1.000 7.86 ATOM 709 CB ASP 95 −32.522 −49.91331.880 1.000 5.49 ATOM 710 CG ASP 95 −32.090 −50.392 30.521 1.000 10.09ATOM 711 OD1 ASP 95 −30.998 −50.021 30.040 1.000 16.22 ATOM 712 OD2 ASP95 −32.843 −51.184 29.907 1.000 15.98 ATOM 713 C ASP 95 −33.020 −48.20833.591 1.000 9.17 ATOM 714 O ASP 95 −34.188 −48.361 33.958 1.000 0.43ATOM 715 N THR 96 −32.051 −47.882 34.421 1.000 11.45 ATOM 716 CA THR 96−32.122 −47.529 35.823 1.000 16.75 ATOM 717 CB THR 96 −30.697 −47.63836.412 1.000 24.78 ATOM 718 OG1 THR 96 −30.607 −48.784 37.274 1.00017.62 ATOM 719 CG2 THR 96 −30.350 −46.409 37.229 1.000 12.12 ATOM 720 CTHR 96 −32.697 −46.132 35.997 1.000 12.12 ATOM 721 O THR 96 −33.047−45.678 37.088 1.000 10.94 ATOM 722 N LYS 97 −32.820 −45.406 34.8831.000 12.18 ATOM 723 CA LYS 97 −33.387 −44.060 34.954 1.000 14.27 ATOM724 CB LYS 97 −33.247 −43.336 33.620 1.000 13.25 ATOM 725 CG LYS 97−31.996 −42.477 33.500 1.000 11.50 ATOM 726 CD LYS 97 −31.819 −41.93532.086 1.000 3.08 ATOM 727 CE LYS 97 −30.344 −41.856 31.717 1.000 0.00ATOM 728 NZ LYS 97 −30.131 −41.152 30.416 1.000 0.00 ATOM 729 C LYS 97−34.848 −44.112 35.403 1.000 12.44 ATOM 730 O LYS 97 −35.636 −44.91434.911 1.000 8.04 ATOM 731 N ALA 98 −35.179 −43.246 36.355 1.000 11.97ATOM 732 CA ALA 98 −36.454 −43.218 37.047 1.000 4.97 ATOM 733 CB ALA 98−36.522 −41.982 37.943 1.000 3.36 ATOM 734 C ALA 98 −37.641 −43.24636.100 1.000 12.00 ATOM 735 O ALA 98 −38.651 −43.905 36.355 1.000 22.61ATOM 736 N TYR 99 −37.535 −42.518 34.988 1.000 12.39 ATOM 737 CA TYR 99−38.695 −42.403 34.107 1.000 7.25 ATOM 738 CB TYR 99 −38.521 −41.29733.087 1.000 9.11 ATOM 739 CG TYR 99 −37.300 −41.251 32.217 1.000 15.58ATOM 740 CD1 TYR 99 −37.261 −41.912 30.995 1.000 13.09 ATOM 741 CE1 TYR99 −36.144 −41.874 30.186 1.000 9.06 ATOM 742 CD2 TYR 99 −36.173 −40.53332.598 1.000 14.48 ATOM 743 CE2 TYR 99 −35.051 −40.482 31.796 1.00015.13 ATOM 744 CZ TYR 99 −35.044 −41.154 30.591 1.000 11.74 ATOM 745 OHTYR 99 −33.925 −41.102 29.794 1.000 6.20 ATOM 746 C TYR 99 −38.990−43.726 33.413 1.000 11.25 ATOM 747 O TYR 99 −40.121 −43.927 32.9631.000 12.89 ATOM 748 N PHE 100 −37.993 −44.606 33.351 1.000 4.63 ATOM749 CA PHE 100 −38.237 −45.908 32.731 1.000 1.01 ATOM 750 CB PHE 100−36.903 −46.556 32.348 1.000 3.41 ATOM 751 CG PHE 100 −36.316 −45.98031.070 1.000 11.77 ATOM 752 CD1 PHE 100 −35.018 −45.506 31.032 1.0007.50 ATOM 753 CD2 PHE 100 −37.080 −45.919 29.917 1.000 16.94 ATOM 754CE1 PHE 100 −34.489 −44.981 29.868 1.000 7.31 ATOM 755 CE2 PHE 100−36.557 −45.398 28.748 1.000 12.92 ATOM 756 CZ PHE 100 −35.260 −44.92528.722 1.000 7.58 ATOM 757 C PHE 100 −39.051 −46.829 33.628 1.000 6.94ATOM 758 O PHE 100 −39.711 −47.750 33.131 1.000 9.31 ATOM 759 N ARG 101−39.032 −46.629 34.943 1.000 12.10 ATOM 760 CA ARG 101 −39.783 −47.46835.869 1.000 12.96 ATOM 761 CB ARG 101 −41.294 −47.296 35.695 1.00016.21 ATOM 762 CG ARG 101 −41.890 −45.959 36.087 1.000 19.51 ATOM 763 CDARG 101 −43.376 −45.918 35.740 1.000 25.82 ATOM 764 NE ARG 101 −43.818−44.553 35.466 1.000 31.88 ATOM 765 CZ ARG 101 −43.797 −43.583 36.3731.000 33.97 ATOM 766 NH1 ARG 101 −43.355 −43.839 37.599 1.000 43.49 ATOM767 NH2 ARG 101 −44.206 −42.361 36.067 1.000 44.85 ATOM 768 C ARG 101−39.472 −48.955 35.704 1.000 12.20 ATOM 769 O ARG 101 −40.376 −49.78235.878 1.000 12.48 ATOM 770 N ARG 102 −38.238 −49.319 35.378 1.000 8.86ATOM 771 CA ARG 102 −37.887 −50.733 35.264 1.000 11.00 ATOM 772 CB ARG102 −36.899 −50.962 34.115 1.000 6.96 ATOM 773 CG ARG 102 −37.497−50.805 32.720 1.000 9.64 ATOM 774 CD ARG 102 −36.518 −51.198 31.6241.000 8.07 ATOM 775 NE ARG 102 −37.140 −51.842 30.474 1.000 4.64 ATOM776 CZ ARG 102 −36.540 −52.606 29.571 1.000 7.34 ATOM 777 NH1 ARG 102−35.240 −52.877 29.628 1.000 1.45 ATOM 778 NH2 ARG 102 −37.232 −53.13128.567 1.000 6.11 ATOM 779 C ARG 102 −37.320 −51.275 36.577 1.000 11.09ATOM 780 O ARG 102 −36.734 −50.567 37.394 1.000 10.02 ATOM 781 N THR 103−37.497 −52.573 36.785 1.000 11.01 ATOM 782 CA THR 103 −36.898 −53.30737.893 1.000 12.65 ATOM 783 CB THR 103 −37.844 −54.376 38.462 1.000 7.64ATOM 784 OG1 THR 103 −38.083 −55.384 37.468 1.000 11.29 ATOM 785 CG2 THR103 −39.199 −53.771 38.790 1.000 15.33 ATOM 786 C THR 103 −35.618−53.966 37.390 1.000 10.55 ATOM 787 O THR 103 −35.409 −53.986 36.1731.000 9.17 ATOM 788 N PRO 104 −34.765 −54.474 38.264 1.000 10.17 ATOM789 CD PRO 104 −34.799 −54.363 39.731 1.000 14.03 ATOM 790 CA PRO 104−33.598 −55.230 37.803 1.000 6.81 ATOM 791 CB PRO 104 −32.968 −55.74839.094 1.000 5.25 ATOM 792 CG PRO 104 −33.402 −54.759 40.129 1.000 8.07ATOM 793 C PRO 104 −34.010 −56.400 36.911 1.000 5.89 ATOM 794 O PRO 104−33.251 −56.728 35.998 1.000 5.49 ATOM 795 N LEU 105 −35.164 −56.99437.173 1.000 2.55 ATOM 796 CA LEU 105 −35.690 −58.071 36.341 1.000 10.27ATOM 797 CB LEU 105 −36.989 −58.642 36.890 1.000 11.51 ATOM 798 CG LEU105 −37.304 −60.122 36.695 1.000 16.39 ATOM 799 CD1 LEU 105 −38.804−60.319 36.480 1.000 4.05 ATOM 800 CD2 LEU 105 −36.533 −60.744 35.5421.000 15.49 ATOM 801 C LEU 105 −35.923 −57.566 34.915 1.000 14.30 ATOM802 O LEU 105 −35.415 −58.168 33.969 1.000 14.22 ATOM 803 N ASP 106−36.686 −56.484 34.791 1.000 11.11 ATOM 804 CA ASP 106 −36.922 −55.87833.482 1.000 8.08 ATOM 805 CB ASP 106 −37.636 −54.538 33.621 1.000 14.02ATOM 806 CG ASP 106 −39.046 −54.638 34.152 1.000 13.88 ATOM 807 OD1 ASP106 −39.726 −55.653 33.875 1.000 19.94 ATOM 808 OD2 ASP 106 −39.479−53.686 34.843 1.000 4.29 ATOM 809 C ASP 106 −35.607 −55.668 32.7341.000 7.79 ATOM 810 O ASP 106 −35.504 −55.987 31.554 1.000 10.52 ATOM811 N ILE 107 −34.614 −55.131 33.438 1.000 5.00 ATOM 812 CA ILE 107−33.321 −54.814 32.845 1.000 6.63 ATOM 813 CB ILE 107 −32.444 −54.01633.828 1.000 14.49 ATOM 814 CG2 ILE 107 −31.125 −53.622 33.184 1.0007.24 ATOM 815 CG1 ILE 107 −33.146 −52.790 34.415 1.000 16.93 ATOM 816CD1 ILE 107 −32.174 −51.779 34.992 1.000 19.38 ATOM 817 C ILE 107−32.564 −56.059 32.405 1.000 5.12 ATOM 818 O ILE 107 −31.877 −56.02431.381 1.000 4.80 ATOM 819 N ALA 108 −32.691 −57.148 33.157 1.000 5.34ATOM 820 CA ALA 108 −32.021 −58.398 32.812 1.000 4.25 ATOM 821 CB ALA108 −32.089 −59.399 33.956 1.000 2.49 ATOM 822 C ALA 108 −32.637 −59.01831.568 1.000 2.89 ATOM 823 O ALA 108 −31.952 −59.619 30.738 1.000 11.68ATOM 824 N LEU 109 −33.956 −58.864 31.449 1.000 0.00 ATOM 825 CA LEU 109−34.609 −59.401 30.251 1.000 6.18 ATOM 826 CB LEU 109 −36.125 −59.39130.435 1.000 12.37 ATOM 827 CG LEU 109 −36.674 −60.463 31.386 1.00015.66 ATOM 828 CD1 LEU 109 −37.985 −60.004 32.001 1.000 27.44 ATOM 829CD2 LEU 109 −36.854 −61.794 30.672 1.000 3.14 ATOM 830 C LEU 109 −34.171−58.620 29.022 1.000 10.30 ATOM 831 O LEU 109 −34.035 −59.139 27.9151.000 18.00 ATOM 832 N GLY 110 −33.918 −57.323 29.193 1.000 11.78 ATOM833 CA GLY 110 −33.426 −56.535 28.069 1.000 8.26 ATOM 834 C GLY 110−32.028 −56.976 27.666 1.000 7.06 ATOM 835 O GLY 110 −31.757 −57.15526.482 1.000 18.68 ATOM 836 N MET 111 −31.149 −57.149 28.651 1.000 5.04ATOM 837 CA MET 111 −29.812 −57.661 28.414 1.000 4.52 ATOM 838 CB MET111 −28.962 −57.717 29.683 1.000 1.61 ATOM 839 CG MET 111 −27.663−58.503 29.542 1.000 0.00 ATOM 840 XD MET 111 −26.456 −57.694 28.4531.000 16.83 ATOM 841 CE MET 111 −25.895 −56.355 29.497 1.000 5.08 ATOM842 C MET 111 −29.915 −59.066 27.821 1.000 6.40 ATOM 843 O MET 111−29.098 −59.476 27.005 1.000 8.66 ATOM 844 N SER 112 −30.937 −59.79528.270 1.000 9.55 ATOM 845 CA SER 112 −31.140 −61.133 27.731 1.000 8.05ATOM 846 CB SER 112 −32.322 −61.821 28.405 1.000 10.37 ATOM 847 OG SER112 −33.488 −61.744 27.609 1.000 8.11 ATOM 848 C SER 112 −31.341 −61.03426.217 1.000 6.07 ATOM 849 O SER 112 −30.761 −61.823 25.471 1.000 9.26ATOM 850 N VAL 113 −32.142 −60.065 25.803 1.000 4.80 ATOM 851 CA VAL 113−32.424 −59.788 24.401 1.000 9.22 ATOM 852 CB VAL 113 −33.414 −58.61524.266 1.000 9.35 ATOM 853 CG1 VAL 113 −33.350 −57.979 22.886 1.000 0.53ATOM 854 CG2 VAL 113 −34.830 −59.090 24.567 1.000 15.43 ATOM 855 C VAL113 −31.149 −59.490 23.616 1.000 18.19 ATOM 856 O VAL 113 −31.027−59.900 22.456 1.000 17.08 ATOM 857 N LEU 114 −30.199 −58.791 24.2351.000 16.22 ATOM 858 CA LEU 114 −28.948 −58.431 23.570 1.000 9.05 ATOM859 CB LEU 114 −28.220 −57.329 24.341 1.000 4.93 ATOM 860 CG LEU 114−28.938 −55.983 24.427 1.000 6.23 ATOM 861 CD1 LEU 114 −28.122 −54.97325.221 1.000 8.47 ATOM 862 CD2 LEU 114 −29.228 −55.450 23.032 1.000 0.00ATOM 863 C LEU 114 −28.018 −59.628 23.407 1.000 5.15 ATOM 864 O LEU 114−27.310 −59.762 22.410 1.000 8.05 ATOM 865 N VAL 115 −28.028 −60.50324.403 1.000 5.78 ATOM 866 CA VAL 115 −27.223 −61.717 24.373 1.000 8.93ATOM 867 CB VAL 115 −27.202 −62.383 25.762 1.000 8.05 ATOM 868 CG1 VAL115 −26.501 −63.729 25.720 1.000 0.00 ATOM 869 CG2 VAL 115 −26.543−61.439 26.759 1.000 0.00 ATOM 870 C VAL 115 −27.763 −62.685 23.3301.000 9.50 ATOM 871 O VAL 115 −27.007 −63.390 22.662 1.000 9.58 ATOM 872N THR 116 −29.087 −62.715 23.179 1.000 8.15 ATOM 873 CA THR 116 −29.688−63.617 22.199 1.000 8.38 ATOM 874 CB THR 116 −31.222 −63.622 22.3271.000 12.50 ATOM 875 OG1 THR 116 −31.575 −64.207 23.585 1.000 13.40 ATOM876 CG2 THR 116 −31.848 −64.479 21.233 1.000 10.82 ATOM 877 C THR 116−29.316 −63.241 20.771 1.000 5.56 ATOM 878 O THR 116 −29.011 −64.12719.966 1.000 5.27 ATOM 879 N GLN 117 −29.345 −61.945 20.473 1.000 8.17ATOM 880 CA GLN 117 −28.956 −61.430 19.160 1.000 9.93 ATOM 881 CB GLN117 −29.166 −59.920 19.080 1.000 3.66 ATOM 882 CG GLN 117 −30.592−59.440 19.279 1.000 6.21 ATOM 883 CD GLN 117 −30.699 −57.933 19.3901.000 7.09 ATOM 884 OE1 GLN 117 −29.801 −57.260 19.896 1.000 12.85 ATOM885 NE2 GLN 117 −31.811 −57.376 18.914 1.000 7.39 ATOM 886 C GLN 117−27.499 −61.761 18.847 1.000 11.60 ATOM 887 O GLN 117 −27.105 −62.02317.706 1.000 9.03 ATOM 888 N VAL 118 −26.652 −61.751 19.879 1.000 11.77ATOM 889 CA VAL 118 −25.258 −62.146 19.659 1.000 8.34 ATOM 890 CB VAL118 −24.340 −61.768 20.831 1.000 0.49 ATOM 891 CG1 VAL 118 −22.892−62.118 20.499 1.000 21.94 ATOM 892 CG2 VAL 118 −24.452 −60.291 21.1691.000 3.31 ATOM 893 C VAL 118 −25.166 −63.652 19.417 1.000 10.48 ATOM894 O VAL 118 −24.354 −64.107 18.607 1.000 10.54 ATOM 895 N LEU 119−25.993 −64.431 20.112 1.000 7.97 ATOM 896 CA LEU 119 −25.916 −65.88519.993 1.000 8.73 ATOM 897 CB LEU 119 −26.679 −66.572 21.135 1.000 8.06ATOM 898 CG LEU 119 −25.981 −66.556 22.498 1.000 21.06 ATOM 899 CD1 LEU119 −26.800 −67.296 23.548 1.000 5.53 ATOM 900 CD2 LEU 119 −24.580−67.150 22.403 1.000 21.96 ATOM 901 C LEU 119 −26.446 −66.362 18.6491.000 5.78 ATOM 902 O LEU 119 −26.022 −67.409 18.153 1.000 14.06 ATOM903 N THR 120 −27.364 −65.608 18.053 1.000 8.82 ATOM 904 CA THR 120−27.964 −65.985 16.780 1.000 0.00 ATOM 905 CB THR 120 −29.497 −65.79816.815 1.000 6.15 ATOM 906 OG1 THR 120 −29.805 −64.405 16.969 1.00010.14 ATOM 907 CG2 THR 120 −30.121 −66.535 17.994 1.000 0.76 ATOM 908 CTHR 120 −27.419 −65.198 15.594 1.000 10.30 ATOM 909 O THR 120 −28.061−65.190 14.537 1.000 13.46 ATOM 910 N SER 121 −26.272 −64.533 15.7001.000 11.26 ATOM 911 CA SER 121 −25.774 −63.675 14.636 1.000 7.70 ATOM912 CB SER 121 −25.000 −62.487 15.240 1.000 5.36 ATOM 913 OG SER 121−23.826 −62.954 15.886 1.000 3.70 ATOM 914 C SER 121 −24.852 −64.35313.629 1.000 7.89 ATOM 915 O SER 121 −24.360 −63.660 12.730 1.000 13.24ATOM 916 N ALA 122 −24.603 −65.645 13.755 1.000 11.50 ATOM 917 CA ALA122 −23.748 −66.370 12.820 1.000 12.48 ATOM 918 CB ALA 122 −23.820−67.868 13.098 1.000 3.73 ATOM 919 C ALA 122 −24.124 −66.083 11.3701.000 7.92 ATOM 920 O ALA 122 −25.311 −66.050 11.042 1.000 8.42 ATOM 921N GLY 123 −23.125 −65.859 10.529 1.000 7.14 ATOM 922 CA GLY 123 −23.316−65.625 9.115 1.000 3.98 ATOM 923 C GLY 123 −23.643 −64.196 8.735 1.00012.34 ATOM 924 O GLY 123 −23.445 −63.822 7.571 1.000 1.55 ATOM 925 N GLY124 −24.132 −63.404 9.683 1.000 19.09 ATOM 926 CA GLY 124 −24.506−62.016 9.471 1.000 13.26 ATOM 927 C GLY 124 −25.277 −61.809 8.186 1.00010.25 ATOM 928 O GLY 124 −26.403 −62.278 8.018 1.000 10.97 ATOM 929 NVAL 125 −24.684 −61.110 7.217 1.000 12.50 ATOM 930 CA VAL 125 −25.365−60.956 5.930 1.000 9.40 ATOM 931 CB VAL 125 −25.557 −59.477 5.559 1.00014.11 ATOM 932 CG1 VAL 125 −26.156 −59.326 4.168 1.000 13.51 ATOM 933CG2 VAL 125 −26.455 −58.786 6.578 1.000 22.31 ATOM 934 C VAL 125 −24.588−61.675 4.833 1.000 6.71 ATOM 935 O VAL 125 −23.580 −61.151 4.368 1.0004.54 ATOM 936 N GLY 126 −25.047 −62.850 4.427 1.000 14.20 ATOM 937 CAGLY 126 −24.466 −63.654 3.377 1.000 9.15 ATOM 938 C GLY 126 −23.012−64.018 3.580 1.000 10.06 ATOM 939 O GLY 126 −22.225 −64.068 2.629 1.0004.29 ATOM 940 N THR 127 −22.595 −64.295 4.811 1.000 6.29 ATOM 941 CA THR127 −21.214 −64.701 5.050 1.000 3.83 ATOM 942 CB THR 127 −20.470 −63.7075.957 1.000 8.35 ATOM 943 OG1 THR 127 −20.719 −64.001 7.339 1.000 16.55ATOM 944 CG2 THR 127 −20.987 −62.295 5.716 1.000 11.34 ATOM 945 C THR127 −21.143 −66.099 5.663 1.000 1.10 ATOM 946 O THR 127 −22.159 −66.6996.001 1.000 4.52 ATOM 947 N THR 128 −19.921 −66.590 5.790 1.000 9.21ATOM 948 CA THR 128 −19.546 −67.893 6.299 1.000 8.72 ATOM 949 CB THR 128−18.451 −68.505 5.397 1.000 10.99 ATOM 950 OG1 THR 128 −17.447 −67.4975.236 1.000 7.85 ATOM 951 CG2 THR 128 −18.976 −68.853 4.015 1.000 3.45ATOM 952 C THR 128 −18.995 −67.821 7.718 1.000 13.03 ATOM 953 O THR 128−18.450 −68.788 8.255 1.000 8.50 ATOM 954 N TYR 129 −19.127 −66.6468.315 1.000 10.20 ATOM 955 CA TYR 129 −18.542 −66.357 9.615 1.000 7.58ATOM 956 CB TYR 129 −18.323 −64.853 9.722 1.000 8.22 ATOM 957 CG TYR 129−17.246 −64.280 8.835 1.000 11.97 ATOM 958 CD1 TYR 129 −17.514 −63.1768.031 1.000 8.62 ATOM 959 CE1 TYR 129 −16.547 −62.636 7.211 1.000 7.23ATOM 960 CD2 TYR 129 −15.970 −64.827 8.799 1.000 12.10 ATOM 961 CE2 TYR129 −14.991 −64.290 7.982 1.000 16.92 ATOM 962 CZ TYR 129 −15.288−63.196 7.193 1.000 16.10 ATOM 963 OH TYR 129 −14.315 −62.655 6.3831.000 11.56 ATOM 964 C TYR 129 −19.416 −66.840 10.765 1.000 9.63 ATOM965 O TYR 129 −20.644 −66.723 10.714 1.000 13.75 ATOM 966 N PRO 130−18.789 −67.380 11.804 1.000 8.51 ATOM 967 CD PRO 130 −17.336 −67.52312.004 1.000 10.11 ATOM 968 CA PRO 130 −19.549 −67.914 12.938 1.000 5.53ATOM 969 CB PRO 130 −18.522 −68.804 13.647 1.000 8.51 ATOM 970 CG PRO130 −17.227 −68.097 13.397 1.000 11.17 ATOM 971 C PRO 130 −19.983−66.791 13.872 1.000 7.77 ATOM 972 O PRO 130 −19.500 −65.667 13.7301.000 2.72 ATOM 973 N ALA 131 −20.873 −67.117 14.799 1.000 7.61 ATOM 974CA ALA 131 −21.305 −66.205 15.844 1.000 2.73 ATOM 975 CB ALA 131 −22.537−66.747 16.554 1.000 0.00 ATOM 976 C ALA 131 −20.174 −65.984 16.8421.000 8.30 ATOM 977 O ALA 131 −19.502 −66.942 17.223 1.000 12.18 ATOM978 N PRO 132 −19.937 −64.752 17.273 1.000 14.28 ATOM 979 CD PRO 132−20.610 −63.516 16.842 1.000 11.04 ATOM 980 CA PRO 132 −18.901 −64.50518.284 1.000 12.37 ATOM 981 CB PRO 132 −18.696 −62.992 18.181 1.00014.35 ATOM 982 CG PRO 132 −20.032 −62.472 17.753 1.000 12.70 ATOM 983 CPRO 132 −19.395 −64.884 19.675 1.000 12.80 ATOM 984 O PRO 132 −20.608−65.027 19.856 1.000 21.24 ATOM 985 N LYS 133 −18.497 −65.051 20.6411.000 14.17 ATOM 986 CA LYS 133 −18.903 −65.337 22.017 1.000 14.31 ATOM987 CB LYS 133 −17.760 −65.881 22.869 1.000 14.22 ATOM 988 CG LYS 133−17.050 −67.101 22.317 1.000 13.51 ATOM 989 CD LYS 133 −15.746 −67.35823.057 1.000 18.76 ATOM 990 CE LYS 133 −15.463 −68.849 23.174 1.00021.23 ATOM 991 NZ LYS 133 −15.154 −69.237 24.580 1.000 37.08 ATOM 992 CLYS 133 −19.441 −64.066 22.667 1.000 10.23 ATOM 993 O LYS 133 −19.319−62.982 22.091 1.000 4.45 ATOM 994 N VAL 134 −20.032 −64.194 23.8531.000 4.74 ATOM 995 CA VAL 134 −20.562 −63.000 24.507 1.000 10.55 ATOM996 CB VAL 134 −22.106 −62.964 24.490 1.000 11.86 ATOM 997 CG1 VAL 134−22.586 −61.523 24.423 1.000 0.00 ATOM 998 CG2 VAL 134 −22.659 −63.77823.334 1.000 29.88 ATOM 999 C VAL 134 −20.129 −62.885 25.963 1.000 12.01ATOM 1000 O VAL 134 −20.215 −63.837 26.736 1.000 27.94 ATOM 1001 N LEU135 −19.676 −61.703 26.357 1.000 12.21 ATOM 1002 CA LEU 135 −19.364−61.443 27.757 1.000 14.41 ATOM 1003 CB LEU 135 −17.975 −60.835 27.8981.000 17.37 ATOM 1004 CG LEU 135 −17.123 −61.223 29.105 1.000 18.57 ATOM1005 CD1 LEU 135 −15.993 −60.213 29.264 1.000 4.42 ATOM 1006 CD2 LEU 135−17.932 −61.341 30.387 1.000 6.01 ATOM 1007 C LEU 135 −20.397 −60.49728.360 1.000 17.03 ATOM 1008 O LEU 135 −20.485 −59.326 27.984 1.00014.19 ATOM 1009 N VAL 136 −21.196 −60.988 29.303 1.000 19.10 ATOM 1010CA VAL 136 −22.167 −60.110 29.954 1.000 14.45 ATOM 1011 CB VAL 136−23.344 −60.925 30.511 1.000 13.65 ATOM 1012 CG1 VAL 136 −24.272 −60.04531.335 1.000 8.06 ATOM 1013 CG2 VAL 136 −24.080 −61.596 29.362 1.0000.00 ATOM 1014 C VAL 136 −21.498 −59.327 31.073 1.000 10.63 ATOM 1015 OVAL 136 −20.929 −59.948 31.971 1.000 7.12 ATOM 1016 N VAL 137 −21.556−57.997 31.027 1.000 7.93 ATOM 1017 CA VAL 137 −20.882 −57.215 32.0561.000 6.63 ATOM 1018 CB VAL 137 −19.699 −56.397 31.497 1.000 6.08 ATOM1019 CG1 VAL 137 −19.115 −55.512 32.595 1.000 6.59 ATOM 1020 CG2 VAL 137−18.609 −57.291 30.936 1.000 10.34 ATOM 1021 C VAL 137 −21.828 −56.25532.775 1.000 6.02 ATOM 1022 O VAL 137 −22.319 −55.273 32.219 1.000 11.10ATOM 1023 N SER 138 −22.061 −56.558 34.040 1.000 6.05 ATOM 1024 CA SER138 −22.800 −55.715 34.972 1.000 9.77 ATOM 1025 CB SER 138 −23.139−56.523 36.223 1.000 16.98 ATOM 1026 OG SER 138 −23.850 −55.804 37.2021.000 19.18 ATOM 1027 C SER 138 −21.944 −54.496 35.276 1.000 8.41 ATOM1028 O SER 138 −20.779 −54.646 35.652 1.000 13.52 ATOM 1029 N PRO 139−22.459 −53.287 35.096 1.000 12.22 ATOM 1030 CD PRO 139 −23.803 −52.95234.599 1.000 11.54 ATOM 1031 CA PRO 139 −21.657 −52.087 35.389 1.0006.14 ATOM 1032 CB PRO 139 −22.422 −51.015 34.608 1.000 7.78 ATOM 1033 CGPRO 139 −23.848 −51.455 34.731 1.000 3.74 ATOM 1034 C PRO 139 −21.620−51.775 36.875 1.000 3.92 ATOM 1035 O PRO 139 −22.460 −52.217 37.6641.000 10.47 ATOM 1036 N PRO 140 −20.636 −51.014 37.347 1.000 8.52 ATOM1037 CD PRO 140 −19.524 −50.412 36.611 1.000 3.33 ATOM 1038 CA PRO 140−20.591 −50.724 38.788 1.000 13.50 ATOM 1039 CB PRO 140 −19.251 −50.01238.971 1.000 12.27 ATOM 1040 CG PRO 140 −18.843 −49.543 37.623 1.0006.73 ATOM 1041 C PRO 140 −21.748 −49.832 39.228 1.000 15.77 ATOM 1042 OPRO 140 −22.321 −49.073 38.445 1.000 21.96 ATOM 1043 N PRO 141 −22.103−49.939 40.505 1.000 4.93 ATOM 1044 CD PRO 141 −21.487 −50.799 41.5281.000 0.26 ATOM 1045 CA PRO 141 −23.230 −49.172 41.036 1.000 3.17 ATOM1046 CB PRO 141 −23.254 −49.560 42.521 1.000 4.18 ATOM 1047 CG PRO 141−22.591 −50.897 42.556 1.000 0.00 ATOM 1048 C PRO 141 −23.014 −47.67140.890 1.000 10.32 ATOM 1049 O PRO 141 −21.876 −47.203 40.900 1.00017.58 ATOM 1050 N LEU 142 −24.120 −46.942 40.760 1.000 9.20 ATOM 1051 CALEU 142 −24.079 −45.490 40.729 1.000 7.44 ATOM 1052 CB LEU 142 −25.421−44.900 40.288 1.000 7.55 ATOM 1053 CG LEU 142 −25.775 −45.119 38.8121.000 13.23 ATOM 1054 CD1 LEU 142 −27.262 −44.901 38.566 1.000 0.00 ATOM1055 CD2 LEU 142 −24.932 −44.218 37.921 1.000 1.85 ATOM 1056 C LEU 142−23.711 −44.945 42.109 1.000 13.38 ATOM 1057 O LEU 142 −23.764 −45.68043.099 1.000 20.55 ATOM 1058 N ALA 143 −23.363 −43.670 42.126 1.00015.81 ATOM 1059 CA ALA 143 −22.960 −42.941 43.322 1.000 13.69 ATOM 1060CB ALA 143 −21.461 −42.676 43.239 1.000 3.16 ATOM 1061 C ALA 143 −23.762−41.656 43.475 1.000 16.69 ATOM 1062 O ALA 143 −24.500 −41.280 42.5521.000 10.61 ATOM 1063 N PRO 144 −23.668 −40.968 44.609 1.000 19.19 ATOM1064 CD PRO 144 −22.997 −41.377 45.852 1.000 16.93 ATOM 1065 CA PRO 144−24.315 −39.659 44.745 1.000 19.29 ATOM 1066 CB PRO 144 −23.730 −39.07646.031 1.000 17.13 ATOM 1067 CG PRO 144 −22.904 −40.130 46.664 1.00012.97 ATOM 1068 C PRO 144 −24.009 −38.723 43.578 1.000 17.14 ATOM 1069 OPRO 144 −22.902 −38.626 43.048 1.000 12.89 ATOM 1070 N MET 145 −25.049−38.002 43.161 1.000 18.09 ATOM 1071 CA MET 145 −24.925 −37.064 42.0521.000 14.70 ATOM 1072 CB MET 145 −25.912 −37.398 40.942 1.000 21.06 ATOM1073 CG MET 145 −25.711 −38.740 40.263 1.000 24.88 ATOM 1074 XD MET 145−27.259 −39.577 39.860 1.000 18.47 ATOM 1075 CE MET 145 −27.956 −39.80441.495 1.000 34.91 ATOM 1076 C MET 145 −25.155 −35.645 42.559 1.00011.49 ATOM 1077 O MET 145 −26.205 −35.342 43.116 1.000 18.46 ATOM 1078 NPRO 146 −24.182 −34.763 42.367 1.000 6.41 ATOM 1079 CD PRO 146 −22.909−34.993 41.683 1.000 8.62 ATOM 1080 CA PRO 146 −24.325 −33.388 42.8511.000 10.88 ATOM 1081 CB PRO 146 −22.916 −32.814 42.759 1.000 10.59 ATOM1082 CG PRO 146 −22.064 −33.819 42.072 1.000 12.17 ATOM 1083 C PRO 146−25.292 −32.588 41.972 1.000 13.13 ATOM 1084 O PRO 146 −25.999 −31.71242.484 1.000 17.39 ATOM 1085 N HIS 147 −25.311 −32.901 40.677 1.00010.50 ATOM 1086 CA HIS 147 −26.203 −32.215 39.758 1.000 9.69 ATOM 1087CB HIS 147 −25.865 −32.480 38.279 1.000 14.24 ATOM 1088 CG HIS 147−26.441 −31.373 37.431 1.000 6.69 ATOM 1089 CD2 HIS 147 −25.875 −30.29736.850 1.000 5.99 ATOM 1090 ND1 HIS 147 −27.780 −31.296 37.134 1.00011.40 ATOM 1091 CE1 HIS 147 −28.018 −30.226 36.391 1.000 11.68 ATOM 1092NE2 HIS 147 −26.871 −29.600 36.201 1.000 12.68 ATOM 1093 C HIS 147−27.658 −32.596 40.013 1.000 5.47 ATOM 1094 O HIS 147 −28.052 −33.76139.960 1.000 11.15 ATOM 1095 N PRO 148 −28.463 −31.575 40.291 1.00012.88 ATOM 1096 CD PRO 148 −28.098 −30.148 40.322 1.000 12.98 ATOM 1097CA PRO 148 −29.877 −31.806 40.602 1.000 13.30 ATOM 1098 CB PRO 148−30.440 −30.401 40.811 1.000 14.82 ATOM 1099 CG PRO 148 −29.426 −29.45540.267 1.000 16.64 ATOM 1100 C PRO 148 −30.600 −32.508 39.456 1.00015.39 ATOM 1101 O PRO 148 −31.525 −33.290 39.689 1.000 15.71 ATOM 1102 NTRP 149 −30.218 −32.263 38.201 1.000 21.29 ATOM 1103 CA TRP 149 −30.909−32.947 37.109 1.000 15.64 ATOM 1104 CB TRP 149 −30.571 −32.328 35.7501.000 17.31 ATOM 1105 CG TRP 149 −31.296 −33.043 34.639 1.000 10.06 ATOM1106 CD2 TRP 149 −32.715 −33.086 34.444 1.000 4.30 ATOM 1107 CE2 TRP 149−32.952 −33.862 33.295 1.000 8.55 ATOM 1108 CE3 TRP 149 −33.805 −32.54135.129 1.000 4.24 ATOM 1109 CD1 TRP 149 −30.748 −33.774 33.629 1.00011.09 ATOM 1110 NE1 TRP 149 −31.736 −34.272 32.813 1.000 5.61 ATOM 1111CZ2 TRP 149 −34.240 −34.107 32.815 1.000 12.36 ATOM 1112 CZ3 TRP 149−35.076 −32.785 34.654 1.000 13.41 ATOM 1113 CH2 TRP 149 −35.286 −33.56333.505 1.000 14.13 ATOM 1114 C TRP 149 −30.566 −34.432 37.101 1.00012.85 ATOM 1115 O TRP 149 −31.447 −35.290 37.033 1.000 7.92 ATOM 1116 NPHE 150 −29.270 −34.728 37.186 1.000 11.11 ATOM 1117 CA PHE 150 −28.841−36.125 37.305 1.000 11.76 ATOM 1118 CB PHE 150 −27.321 −36.192 37.4831.000 8.65 ATOM 1119 CG PHE 150 −26.581 −36.170 36.150 1.000 13.44 ATOM1120 CD1 PHE 150 −25.315 −35.623 36.047 1.000 14.41 ATOM 1121 CD2 PHE150 −27.167 −36.697 35.014 1.000 12.01 ATOM 1122 CE1 PHE 150 −24.650−35.604 34.838 1.000 14.96 ATOM 1123 CE2 PHE 150 −26.511 −36.684 33.7971.000 13.41 ATOM 1124 CZ PHE 150 −25.246 −36.136 33.711 1.000 18.95 ATOM1125 C PHE 150 −29.555 −36.813 38.459 1.000 10.90 ATOM 1126 O PHE 150−30.059 −37.930 38.354 1.000 7.95 ATOM 1127 N GLN 151 −29.606 −36.12039.598 1.000 12.36 ATOM 1128 CA GLN 151 −30.294 −36.665 40.759 1.00019.45 ATOM 1129 CB GLN 151 −30.306 −35.680 41.932 1.000 12.11 ATOM 1130CG GLN 151 −28.947 −35.446 42.561 1.000 16.34 ATOM 1131 CD GLN 151−29.048 −34.481 43.734 1.000 22.05 ATOM 1132 OE1 GLN 151 −29.693 −34.80344.729 1.000 39.76 ATOM 1133 NE2 GLN 151 −28.423 −33.317 43.598 1.00016.49 ATOM 1134 C GLN 151 −31.745 −37.027 40.441 1.000 20.77 ATOM 1135 OGLN 151 −32.232 −38.044 40.936 1.000 19.36 ATOM 1136 N LEU 152 −32.397−36.183 39.644 1.000 11.67 ATOM 1137 CA LEU 152 −33.818 −36.360 39.3651.000 13.95 ATOM 1138 CB LEU 152 −34.438 −35.101 38.764 1.000 14.14 ATOM1139 CG LEU 152 −34.837 −33.957 39.688 1.000 12.09 ATOM 1140 CD1 LEU 152−34.781 −32.631 38.935 1.000 11.66 ATOM 1141 CD2 LEU 152 −36.225 −34.16240.274 1.000 12.14 ATOM 1142 C LEU 152 −34.053 −37.544 38.428 1.00013.07 ATOM 1143 O LEU 152 −34.913 −38.372 38.729 1.000 13.96 ATOM 1144 NILE 153 −33.310 −37.613 37.326 1.000 13.21 ATOM 1145 CA ILE 153 −33.519−38.661 36.334 1.000 12.12 ATOM 1146 CB ILE 153 −32.814 −38.377 34.9911.000 9.74 ATOM 1147 CG2 ILE 153 −33.360 −37.106 34.355 1.000 0.00 ATOM1148 CG1 ILE 153 −31.284 −38.333 35.061 1.000 8.16 ATOM 1149 CD1 ILE 153−30.635 −38.332 33.684 1.000 0.00 ATOM 1150 C ILE 153 −33.054 −40.02436.836 1.000 9.56 ATOM 1151 O ILE 153 −33.540 −41.043 36.342 1.000 4.79ATOM 1152 N PHE 154 −32.138 −40.069 37.797 1.000 12.41 ATOM 1153 CA PHE154 −31.645 −41.349 38.301 1.000 8.75 ATOM 1154 CB PHE 154 −30.113−41.372 38.348 1.000 8.88 ATOM 1155 CG PHE 154 −29.456 −41.758 37.0311.000 8.38 ATOM 1156 CD1 PHE 154 −28.597 −40.887 36.384 1.000 9.10 ATOM1157 CD2 PHE 154 −29.703 −42.990 36.458 1.000 0.00 ATOM 1158 CE1 PHE 154−28.000 −41.232 35.188 1.000 9.85 ATOM 1159 CE2 PHE 154 −29.119 −43.34435.260 1.000 5.02 ATOM 1160 CZ PHE 154 −28.258 −42.468 34.624 1.000 8.39ATOM 1161 C PHE 154 −32.199 −41.648 39.690 1.000 11.55 ATOM 1162 O PHE154 −31.683 −42.515 40.400 1.000 10.77 ATOM 1163 N GLU 155 −33.246−40.936 40.093 1.000 15.11 ATOM 1164 CA GLU 155 −33.898 −41.221 41.3671.000 19.95 ATOM 1165 CB GLU 155 −35.134 −40.343 41.542 1.000 26.08 ATOM1166 CG GLU 155 −35.558 −40.107 42.980 1.000 33.00 ATOM 1167 CD GLU 155−36.339 −41.267 43.568 1.000 44.51 ATOM 1168 OE1 GLU 155 −37.432 −41.58543.051 1.000 49.47 ATOM 1169 OE2 GLU 155 −35.862 −41.867 44.558 1.00061.39 ATOM 1170 C GLU 155 −34.270 −42.702 41.449 1.000 18.82 ATOM 1171 OGLU 155 −34.978 −43.212 40.582 1.000 14.49 ATOM 1172 N GLY 156 −33.779−43.376 42.481 1.000 12.58 ATOM 1173 CA GLY 156 −33.993 −44.787 42.6961.000 6.50 ATOM 1174 C GLY 156 −33.061 −45.684 41.914 1.000 12.22 ATOM1175 O GLY 156 −33.205 −46.914 41.914 1.000 27.90 ATOM 1176 N GLY 157−32.082 −45.107 41.224 1.000 9.19 ATOM 1177 CA GLY 157 −31.216 −45.87740.358 1.000 8.21 ATOM 1178 C GLY 157 −30.007 −46.514 40.991 1.000 8.61ATOM 1179 O GLY 157 −29.563 −47.579 40.549 1.000 17.22 ATOM 1180 N GLU158 −29.442 −45.887 42.018 1.000 7.58 ATOM 1181 CA GLU 158 −28.299−46.453 42.721 1.000 7.50 ATOM 1182 CB GLU 158 −27.807 −45.505 43.8141.000 9.84 ATOM 1183 CG GLU 158 −26.756 −46.097 44.739 1.000 11.00 ATOM1184 CD GLU 158 −26.031 −45.053 45.564 1.000 24.40 ATOM 1185 OE1 GLU 158−26.158 −43.845 45.267 1.000 33.57 ATOM 1186 OE2 GLU 158 −25.325 −45.43946.523 1.000 39.11 ATOM 1187 C GLU 158 −28.696 −47.807 43.302 1.00013.34 ATOM 1188 O GLU 158 −27.956 −48.787 43.225 1.000 29.78 ATOM 1189 NGLN 159 −29.895 −47.840 43.875 1.000 10.17 ATOM 1190 CA GLN 159 −30.481−49.058 44.406 1.000 15.50 ATOM 1191 CB GLN 159 −31.856 −48.764 45.0171.000 19.57 ATOM 1192 CG GLN 159 −32.548 −49.952 45.647 1.000 24.93 ATOM1193 CD GLN 159 −31.737 −50.676 46.704 1.000 30.24 ATOM 1194 OE1 GLN 159−31.940 −50.499 47.909 1.000 40.80 ATOM 1195 NE2 GLN 159 −30.800 −51.51046.265 1.000 20.75 ATOM 1196 C GLN 159 −30.605 −50.132 43.336 1.00017.89 ATOM 1197 O GLN 159 −30.218 −51.285 43.544 1.000 21.71 ATOM 1198 NLYS 160 −31.154 −49.791 42.168 1.000 15.99 ATOM 1199 CA LYS 160 −31.361−50.855 41.176 1.000 6.75 ATOM 1200 CB LYS 160 −32.314 −50.369 40.0901.000 10.24 ATOM 1201 CG LYS 160 −33.666 −49.907 40.607 1.000 6.13 ATOM1202 CD LYS 160 −34.386 −49.041 39.581 1.000 11.21 ATOM 1203 CE LYS 160−35.897 −49.190 39.702 1.000 9.55 ATOM 1204 NZ LYS 160 −36.616 −48.23538.811 1.000 20.37 ATOM 1205 C LYS 160 −30.029 −51.305 40.591 1.00014.32 ATOM 1206 O LYS 160 −29.842 −52.475 40.257 1.000 14.42 ATOM 1207 NTHR 161 −29.082 −50.375 40.465 1.000 10.29 ATOM 1208 CA THR 161 −27.771−50.734 39.933 1.000 13.43 ATOM 1209 CB THR 161 −26.878 −49.508 39.6721.000 10.03 ATOM 1210 OG1 THR 161 −27.070 −48.557 40.730 1.000 30.01ATOM 1211 CG2 THR 161 −27.263 −48.788 38.389 1.000 13.57 ATOM 1212 C THR161 −27.057 −51.683 40.896 1.000 12.06 ATOM 1213 O THR 161 −26.160−52.415 40.481 1.000 6.51 ATOM 1214 N THR 162 −27.457 −51.664 42.1651.000 8.39 ATOM 1215 CA THR 162 −26.894 −52.551 43.177 1.000 9.75 ATOM1216 CB THR 162 −27.286 −52.130 44.604 1.000 12.96 ATOM 1217 OG1 THR 162−26.705 −50.863 44.941 1.000 11.98 ATOM 1218 CG2 THR 162 −26.735 −53.13245.605 1.000 20.35 ATOM 1219 C THR 162 −27.349 −53.991 42.956 1.00010.87 ATOM 1220 O THR 162 −26.764 −54.942 43.471 1.000 12.87 ATOM 1221 NGLU 163 −28.410 −54.170 42.174 1.000 16.58 ATOM 1222 CA GLU 163 −28.949−55.496 41.905 1.000 20.69 ATOM 1223 CB GLU 163 −30.486 −55.450 41.8611.000 21.36 ATOM 1224 CG GLU 163 −31.136 −54.918 43.122 1.000 19.81 ATOM1225 CD GLU 163 −30.918 −55.799 44.332 1.000 20.57 ATOM 1226 OE1 GLU 163−30.336 −56.894 44.181 1.000 13.38 ATOM 1227 OE2 GLU 163 −31.340 −55.39445.441 1.000 37.36 ATOM 1228 C GLU 163 −28.455 −56.101 40.596 1.00012.31 ATOM 1229 O GLU 163 −28.614 −57.306 40.384 1.000 8.17 ATOM 1230 NLEU 164 −27.880 −55.296 39.710 1.000 14.12 ATOM 1231 CA LEU 164 −27.561−55.746 38.356 1.000 8.92 ATOM 1232 CB LEU 164 −26.960 −54.602 37.5411.000 5.54 ATOM 1233 CG LEU 164 −27.903 −53.857 36.593 1.000 10.39 ATOM1234 CD1 LEU 164 −29.295 −53.740 37.197 1.000 23.43 ATOM 1235 CD2 LEU164 −27.352 −52.485 36.240 1.000 2.48 ATOM 1236 C LEU 164 −26.621−56.943 38.361 1.000 6.54 ATOM 1237 O LEU 164 −26.847 −57.925 37.6531.000 4.26 ATOM 1238 N ALA 165 −25.562 −56.865 39.159 1.000 7.24 ATOM1239 CA ALA 165 −24.609 −57.965 39.239 1.000 11.41 ATOM 1240 CB ALA 165−23.542 −57.659 40.276 1.000 11.40 ATOM 1241 C ALA 165 −25.312 −59.28439.551 1.000 16.26 ATOM 1242 O ALA 165 −24.980 −60.302 38.947 1.00018.13 ATOM 1243 N ARG 166 −26.266 −59.245 40.469 1.000 20.04 ATOM 1244CA ARG 166 −27.014 −60.397 40.947 1.000 10.10 ATOM 1245 CB ARG 166−27.875 −59.992 42.145 1.000 15.40 ATOM 1246 CG ARG 166 −28.600 −61.12742.843 1.000 15.67 ATOM 1247 CD ARG 166 −29.286 −60.640 44.115 1.00020.34 ATOM 1248 NE ARG 166 −30.097 −59.453 43.851 1.000 31.99 ATOM 1249CZ ARG 166 −31.261 −59.505 43.202 1.000 37.46 ATOM 1250 NH1 ARG 166−31.718 −60.673 42.770 1.000 41.26 ATOM 1251 NH2 ARG 166 −31.974 −58.41042.979 1.000 44.85 ATOM 1252 C ARG 166 −27.899 −60.991 39.862 1.00010.33 ATOM 1253 O ARG 166 −27.862 −62.186 39.569 1.000 11.28 ATOM 1254 NVAL 167 −28.724 −60.143 39.253 1.000 10.14 ATOM 1255 CA VAL 167 −29.647−60.637 38.231 1.000 8.08 ATOM 1256 CB VAL 167 −30.800 −59.642 38.0071.000 12.63 ATOM 1257 CG1 VAL 167 −31.873 −60.262 37.129 1.000 23.15ATOM 1258 CG2 VAL 167 −31.423 −59.212 39.331 1.000 16.49 ATOM 1259 C VAL167 −28.941 −60.943 36.916 1.000 8.93 ATOM 1260 O VAL 167 −29.342−61.889 36.230 1.000 11.00 ATOM 1261 N TYR 168 −27.906 −60.209 36.5071.000 6.53 ATOM 1262 CA TYR 168 −27.225 −60.549 35.262 1.000 5.82 ATOM1263 CB TYR 168 −26.220 −59.494 34.815 1.000 12.35 ATOM 1264 CG TYR 168−26.746 −58.249 34.148 1.000 10.53 ATOM 1265 CD1 TYR 168 −25.898 −57.41533.429 1.000 4.25 ATOM 1266 CE1 TYR 168 −26.377 −56.273 32.816 1.0003.59 ATOM 1267 CD2 TYR 168 −28.085 −57.889 34.230 1.000 9.22 ATOM 1268CE2 TYR 168 −28.565 −56.750 33.624 1.000 11.67 ATOM 1269 CZ TYR 168−27.708 −55.940 32.912 1.000 8.76 ATOM 1270 OH TYR 168 −28.194 −54.80132.308 1.000 13.56 ATOM 1271 C TYR 168 −26.466 −61.863 35.444 1.000 9.45ATOM 1272 O TYR 168 −26.398 −62.696 34.544 1.000 5.20 ATOM 1273 N SER169 −25.896 −61.972 36.648 1.000 5.94 ATOM 1274 CA SER 169 −25.145−63.174 36.999 1.000 11.65 ATOM 1275 CB SER 169 −24.663 −63.109 38.4451.000 12.52 ATOM 1276 OG SER 169 −23.611 −64.024 38.688 1.000 13.86 ATOM1277 C SER 169 −26.034 −64.389 36.740 1.000 14.93 ATOM 1278 O SER 169−25.709 −65.240 35.912 1.000 25.35 ATOM 1279 N ALA 170 −27.161 −64.43437.448 1.000 9.54 ATOM 1280 CA ALA 170 −28.154 −65.483 37.259 1.000 7.33ATOM 1281 CB ALA 170 −29.397 −65.155 38.069 1.000 3.12 ATOM 1282 C ALA170 −28.495 −65.659 35.785 1.000 12.27 ATOM 1283 O ALA 170 −28.526−66.772 35.262 1.000 20.56 ATOM 1284 N LEU 171 −28.753 −64.558 35.0811.000 15.11 ATOM 1285 CA LEU 171 −29.115 −64.661 33.665 1.000 17.04 ATOM1286 CB LEU 171 −29.329 −63.272 33.076 1.000 13.64 ATOM 1287 CG LEU 171−29.846 −63.164 31.645 1.000 21.08 ATOM 1288 CD1 LEU 171 −28.692 −63.04330.658 1.000 45.18 ATOM 1289 CD2 LEU 171 −30.734 −64.340 31.270 1.00017.34 ATOM 1290 C LEU 171 −28.052 −65.404 32.868 1.000 18.57 ATOM 1291 OLEU 171 −28.328 −66.409 32.219 1.000 17.64 ATOM 1292 N ALA 172 −26.825−64.890 32.920 1.000 22.46 ATOM 1293 CA ALA 172 −25.735 −65.489 32.1571.000 17.47 ATOM 1294 CB ALA 172 −24.454 −64.699 32.377 1.000 10.29 ATOM1295 C ALA 172 −25.549 −66.953 32.536 1.000 13.15 ATOM 1296 O ALA 172−25.192 −67.797 31.713 1.000 17.25 ATOM 1297 N SER 173 −25.802 −67.24233.809 1.000 11.55 ATOM 1298 CA SER 173 −25.653 −68.595 34.337 1.00015.80 ATOM 1299 CB SER 173 −25.837 −68.578 35.856 1.000 15.14 ATOM 1300OG SER 173 −26.298 −69.837 36.293 1.000 15.66 ATOM 1301 C SER 173−26.640 −69.565 33.691 1.000 10.39 ATOM 1302 O SER 173 −26.263 −70.66733.284 1.000 5.06 ATOM 1303 N PHE 174 −27.882 −69.119 33.601 1.000 6.57ATOM 1304 CA PHE 174 −28.970 −69.778 32.908 1.000 4.04 ATOM 1305 CB PHE174 −30.288 −69.024 33.114 1.000 4.43 ATOM 1306 CG PHE 174 −31.524−69.765 32.626 1.000 3.57 ATOM 1307 CD1 PHE 174 −32.219 −70.606 33.4751.000 0.40 ATOM 1308 CD2 PHE 174 −31.988 −69.615 31.331 1.000 11.71 ATOM1309 CE1 PHE 174 −33.343 −71.281 33.051 1.000 1.63 ATOM 1310 CE2 PHE 174−33.114 −70.285 30.886 1.000 10.57 ATOM 1311 CZ PHE 174 −33.795 −71.11931.756 1.000 10.59 ATOM 1312 C PHE 174 −28.701 −69.872 31.408 1.000 8.80ATOM 1313 O PHE 174 −28.846 −70.949 30.834 1.000 0.14 ATOM 1314 N MET175 −28.328 −68.751 30.793 1.000 7.91 ATOM 1315 CA MET 175 −28.058−68.739 29.356 1.000 5.97 ATOM 1316 CB MET 175 −28.103 −67.321 28.7801.000 0.00 ATOM 1317 CG MET 175 −29.492 −66.712 28.751 1.000 7.42 ATOM1318 XD MET 175 −29.573 −65.056 28.023 1.000 16.37 ATOM 1319 CE MET 175−30.064 −65.488 26.348 1.000 21.02 ATOM 1320 C MET 175 −26.715 −69.39929.045 1.000 6.31 ATOM 1321 O MET 175 −26.332 −69.479 27.880 1.000 8.17ATOM 1322 N LYS 176 −26.020 −69.872 30.070 1.000 8.77 ATOM 1323 CA LYS176 −24.762 −70.598 29.939 1.000 10.68 ATOM 1324 CB LYS 176 −24.970−71.945 29.239 1.000 10.45 ATOM 1325 CG LYS 176 −25.907 −72.900 29.9711.000 3.74 ATOM 1326 CD LYS 176 −25.133 −73.755 30.964 1.000 5.05 ATOM1327 CE LYS 176 −26.084 −74.568 31.833 1.000 6.09 ATOM 1328 NZ LYS 176−26.739 −73.721 32.861 1.000 24.38 ATOM 1329 C LYS 176 −23.733 −69.76029.190 1.000 12.34 ATOM 1330 O LYS 176 −23.084 −70.178 28.231 1.00024.85 ATOM 1331 N VAL 177 −23.601 −68.520 29.648 1.000 12.09 ATOM 1332CA VAL 177 −22.709 −67.581 28.953 1.000 12.10 ATOM 1333 CB VAL 177−23.569 −66.629 28.106 1.000 9.74 ATOM 1334 CG1 VAL 177 −23.831 −65.31928.835 1.000 18.59 ATOM 1335 CG2 VAL 177 −22.921 −66.372 26.753 1.00020.30 ATOM 1336 C VAL 177 −21.848 −66.876 29.982 1.000 13.62 ATOM 1337 OVAL 177 −22.292 −66.730 31.126 1.000 20.25 ATOM 1338 N PRO 178 −20.635−66.454 29.637 1.000 10.56 ATOM 1339 CD PRO 178 −20.019 −66.530 28.3121.000 2.11 ATOM 1340 CA PRO 178 −19.760 −65.842 30.642 1.000 10.32 ATOM1341 CB PRO 178 −18.433 −65.656 29.913 1.000 6.70 ATOM 1342 CG PRO 178−18.623 −66.026 28.499 1.000 0.81 ATOM 1343 C PRO 178 −20.281 −64.48331.119 1.000 20.65 ATOM 1344 O PRO 178 −20.796 −63.674 30.351 1.00022.70 ATOM 1345 N PHE 179 −20.124 −64.253 32.412 1.000 22.55 ATOM 1346CA PHE 179 −20.474 −63.025 33.107 1.000 19.13 ATOM 1347 CB PHE 179−21.518 −63.283 34.194 1.000 8.91 ATOM 1348 CG PHE 179 −21.661 −62.21535.268 1.000 8.12 ATOM 1349 CD1 PHE 179 −22.433 −61.087 35.044 1.00010.36 ATOM 1350 CD2 PHE 179 −21.031 −62.337 36.499 1.000 2.04 ATOM 1351CE1 PHE 179 −22.590 −60.103 36.004 1.000 2.43 ATOM 1352 CE2 PHE 179−21.183 −61.367 37.470 1.000 0.76 ATOM 1353 CZ PHE 179 −21.963 −60.24837.228 1.000 2.96 ATOM 1354 C PHE 179 −19.231 −62.400 33.736 1.000 13.74ATOM 1355 O PHE 179 −18.309 −63.110 34.128 1.000 15.60 ATOM 1356 N PHE180 −19.214 −61.080 33.838 1.000 14.28 ATOM 1357 CA PHE 180 −18.178−60.371 34.573 1.000 13.03 ATOM 1358 CB PHE 180 −17.004 −59.952 33.6861.000 17.94 ATOM 1359 CG PHE 180 −15.933 −59.164 34.433 1.000 21.76 ATOM1360 CD1 PHE 180 −14.960 −59.807 35.176 1.000 21.38 ATOM 1361 CD2 PHE180 −15.904 −57.780 34.391 1.000 19.62 ATOM 1362 CE1 PHE 180 −13.979−59.108 35.859 1.000 15.07 ATOM 1363 CE2 PHE 180 −14.941 −57.064 35.0751.000 21.73 ATOM 1364 CZ PHE 180 −13.979 −57.727 35.816 1.000 21.65 ATOM1365 C PHE 180 −18.822 −59.164 35.256 1.000 12.16 ATOM 1366 O PHE 180−19.594 −58.423 34.648 1.000 11.01 ATOM 1367 N ASP 181 −18.504 −58.98836.536 1.000 7.72 ATOM 1368 CA ASP 181 −19.062 −57.864 37.286 1.00010.61 ATOM 1369 CB ASP 181 −19.521 −58.346 38.659 1.000 5.77 ATOM 1370CG ASP 181 −19.986 −57.225 39.559 1.000 4.11 ATOM 1371 OD1 ASP 181−20.116 −56.076 39.092 1.000 8.61 ATOM 1372 OD2 ASP 181 −20.217 −57.50840.750 1.000 11.49 ATOM 1373 C ASP 181 −18.037 −56.743 37.378 1.00015.44 ATOM 1374 O ASP 181 −17.023 −56.872 38.060 1.000 16.84 ATOM 1375 NALA 182 −18.293 −55.639 36.672 1.000 18.65 ATOM 1376 CA ALA 182 −17.359−54.517 36.678 1.000 18.00 ATOM 1377 CB ALA 182 −17.778 −53.459 35.6681.000 7.66 ATOM 1378 C ALA 182 −17.240 −53.911 38.075 1.000 18.92 ATOM1379 O ALA 182 −16.198 −53.340 38.400 1.000 8.61 ATOM 1380 N GLY 183−18.296 −54.044 38.872 1.000 15.67 ATOM 1381 CA GLY 183 −18.374 −53.51640.219 1.000 13.53 ATOM 1382 C GLY 183 −17.444 −54.230 41.176 1.00014.96 ATOM 1383 O GLY 183 −17.268 −53.846 42.330 1.000 25.31 ATOM 1384 NSER 184 −16.830 −55.306 40.696 1.000 16.38 ATOM 1385 CA SER 184 −15.940−56.105 41.525 1.000 12.32 ATOM 1386 CB SER 184 −16.009 −57.574 41.1161.000 14.55 ATOM 1387 OG SER 184 −15.237 −57.867 39.967 1.000 12.36 ATOM1388 C SER 184 −14.516 −55.572 41.439 1.000 13.33 ATOM 1389 O SER 184−13.644 −55.986 42.204 1.000 12.05 ATOM 1390 N VAL 185 −14.276 −54.64040.515 1.000 9.89 ATOM 1391 CA VAL 185 −12.902 −54.156 40.358 1.00014.54 ATOM 1392 CB VAL 185 −12.320 −54.649 39.021 1.000 16.34 ATOM 1393CG1 VAL 185 −12.034 −56.141 39.100 1.000 13.09 ATOM 1394 CG2 VAL 185−13.274 −54.346 37.877 1.000 20.34 ATOM 1395 C VAL 185 −12.802 −52.64240.445 1.000 20.13 ATOM 1396 O VAL 185 −11.718 −52.101 40.682 1.00011.67 ATOM 1397 N ILE 186 −13.912 −51.929 40.260 1.000 19.83 ATOM 1398CA ILE 186 −13.905 −50.479 40.381 1.000 13.97 ATOM 1399 CB ILE 186−13.716 −49.752 39.031 1.000 8.30 ATOM 1400 CG2 ILE 186 −12.362 −50.07038.428 1.000 12.39 ATOM 1401 CG1 ILE 186 −14.830 −50.005 38.014 1.00010.45 ATOM 1402 CD1 ILE 186 −14.956 −48.929 36.957 1.000 3.60 ATOM 1403C ILE 186 −15.209 −49.957 40.979 1.000 13.38 ATOM 1404 O ILE 186 −16.256−50.583 40.857 1.000 12.90 ATOM 1405 N SER 187 −15.120 −48.788 41.5961.000 11.99 ATOM 1406 CA SER 187 −16.287 −48.046 42.052 1.000 9.16 ATOM1407 CB SER 187 −16.110 −47.594 43.498 1.000 10.88 ATOM 1408 OG SER 187−14.889 −46.879 43.658 1.000 16.58 ATOM 1409 C SER 187 −16.517 −46.83941.145 1.000 11.87 ATOM 1410 O SER 187 −15.567 −46.304 40.563 1.00016.73 ATOM 1411 N THR 188 −17.767 −46.410 41.015 1.000 15.17 ATOM 1412CA THR 188 −18.077 −45.244 40.189 1.000 13.51 ATOM 1413 CB THR 188−19.571 −45.151 39.848 1.000 12.88 ATOM 1414 OG1 THR 188 −19.969 −46.30839.101 1.000 16.33 ATOM 1415 CG2 THR 188 −19.843 −43.943 38.961 1.0008.08 ATOM 1416 C THR 188 −17.639 −43.978 40.916 1.000 14.09 ATOM 1417 OTHR 188 −18.293 −43.535 41.860 1.000 10.72 ATOM 1418 N ASP 189 −16.518−43.414 40.474 1.000 15.51 ATOM 1419 CA ASP 189 −15.911 −42.313 41.2101.000 11.58 ATOM 1420 CB ASP 189 −14.407 −42.594 41.362 1.000 12.86 ATOM1421 CG ASP 189 −14.158 −43.791 42.261 1.000 4.55 ATOM 1422 OD1 ASP 189−14.915 −43.960 43.239 1.000 13.27 ATOM 1423 OD2 ASP 189 −13.208 −44.54941.989 1.000 6.91 ATOM 1424 C ASP 189 −16.120 −40.949 40.567 1.000 15.34ATOM 1425 O ASP 189 −15.910 −39.948 41.263 1.000 18.48 ATOM 1426 N GLY190 −16.510 −40.918 39.303 1.000 19.39 ATOM 1427 CA GLY 190 −16.710−39.718 38.515 1.000 15.08 ATOM 1428 C GLY 190 −17.385 −38.613 39.3031.000 18.57 ATOM 1429 O GLY 190 −18.263 −38.908 40.119 1.000 20.64 ATOM1430 N VAL 191 −16.952 −37.381 39.057 1.000 13.86 ATOM 1431 CA VAL 191−17.428 −36.226 39.806 1.000 10.59 ATOM 1432 CB VAL 191 −16.825 −34.90539.286 1.000 17.05 ATOM 1433 CG1 VAL 191 −15.324 −34.875 39.559 1.00030.84 ATOM 1434 CG2 VAL 191 −17.092 −34.701 37.803 1.000 8.10 ATOM 1435C VAL 191 −18.950 −36.129 39.774 1.000 10.47 ATOM 1436 O VAL 191 −19.542−35.686 40.761 1.000 13.60 ATOM 1437 N ASP 192 −19.571 −36.534 38.6681.000 1.46 ATOM 1438 CA ASP 192 −21.018 −36.447 38.540 1.000 0.70 ATOM1439 CB ASP 192 −21.387 −36.356 37.056 1.000 2.10 ATOM 1440 CG ASP 192−20.918 −37.566 36.268 1.000 9.82 ATOM 1441 OD1 ASP 192 −20.296 −38.47836.857 1.000 8.20 ATOM 1442 OD2 ASP 192 −21.182 −37.597 35.047 1.0006.78 ATOM 1443 C ASP 192 −21.754 −37.622 39.173 1.000 7.73 ATOM 1444 OASP 192 −22.988 −37.674 39.136 1.000 7.10 ATOM 1445 N GLY 193 −21.027−38.572 39.753 1.000 15.10 ATOM 1446 CA GLY 193 −21.631 −39.747 40.3511.000 17.83 ATOM 1447 C GLY 193 −22.153 −40.758 39.352 1.000 18.93 ATOM1448 O GLY 193 −22.820 −41.732 39.718 1.000 10.12 ATOM 1449 N ILE 194−21.867 −40.565 38.062 1.000 11.77 ATOM 1450 CA ILE 194 −22.330 −41.54637.081 1.000 7.87 ATOM 1451 CB ILE 194 −23.401 −40.945 36.154 1.000 9.95ATOM 1452 CG2 ILE 194 −23.790 −41.927 35.063 1.000 0.00 ATOM 1453 CG1ILE 194 −24.643 −40.441 36.896 1.000 9.90 ATOM 1454 CD1 ILE 194 −25.248−39.237 36.206 1.000 8.85 ATOM 1455 C ILE 194 −21.191 −42.068 36.2251.000 2.97 ATOM 1456 O ILE 194 −21.086 −43.251 35.924 1.000 6.72 ATOM1457 N HIS 195 −20.277 −41.195 35.792 1.000 6.33 ATOM 1458 CA HIS 195−19.256 −41.719 34.884 1.000 10.76 ATOM 1459 CB HIS 195 −19.089 −40.79033.673 1.000 11.36 ATOM 1460 CG HIS 195 −20.402 −40.647 32.958 1.00011.50 ATOM 1461 CD2 HIS 195 −20.981 −41.395 31.989 1.000 5.43 ATOM 1462ND1 HIS 195 −21.283 −39.633 33.253 1.000 7.30 ATOM 1463 CE1 HIS 195−22.351 −39.753 32.485 1.000 9.11 ATOM 1464 NE2 HIS 195 −22.192 −40.81431.711 1.000 8.18 ATOM 1465 C HIS 195 −17.918 −41.941 35.577 1.000 8.63ATOM 1466 O HIS 195 −17.762 −41.602 36.743 1.000 13.71 ATOM 1467 N PHE196 −17.010 −42.529 34.812 1.000 6.37 ATOM 1468 CA PHE 196 −15.725−43.017 35.249 1.000 9.06 ATOM 1469 CB PHE 196 −15.233 −44.136 34.3201.000 5.38 ATOM 1470 CG PHE 196 −16.048 −45.412 34.451 1.000 10.20 ATOM1471 CD1 PHE 196 −15.822 −46.481 33.602 1.000 8.01 ATOM 1472 CD2 PHE 196−17.027 −45.509 35.427 1.000 6.21 ATOM 1473 CE1 PHE 196 −16.571 −47.63733.722 1.000 11.17 ATOM 1474 CE2 PHE 196 −17.779 −46.662 35.546 1.00014.06 ATOM 1475 CZ PHE 196 −17.549 −47.727 34.694 1.000 13.03 ATOM 1476C PHE 196 −14.663 −41.925 35.273 1.000 12.92 ATOM 1477 O PHE 196 −14.757−40.983 34.494 1.000 15.16 ATOM 1478 N THR 197 −13.694 −42.112 36.1581.000 13.17 ATOM 1479 CA THR 197 −12.477 −41.318 36.183 1.000 17.95 ATOM1480 CB THR 197 −11.886 −41.168 37.593 1.000 20.94 ATOM 1481 OG1 THR 197−11.650 −42.458 38.173 1.000 20.14 ATOM 1482 CG2 THR 197 −12.882 −40.45438.499 1.000 31.55 ATOM 1483 C THR 197 −11.443 −41.978 35.269 1.00010.26 ATOM 1484 O THR 197 −11.713 −43.037 34.705 1.000 14.53 ATOM 1485 NGLU 198 −10.283 −41.362 35.133 1.000 9.05 ATOM 1486 CA GLU 198 −9.192−41.943 34.362 1.000 12.89 ATOM 1487 CB GLU 198 −8.023 −40.960 34.3141.000 20.40 ATOM 1488 CG GLU 198 −6.903 −41.349 33.362 1.000 32.30 ATOM1489 CD GLU 198 −5.764 −40.346 33.328 1.000 35.77 ATOM 1490 OE1 GLU 198−5.127 −40.141 34.385 1.000 42.59 ATOM 1491 OE2 GLU 198 −5.498 −39.76132.256 1.000 25.40 ATOM 1492 C GLU 198 −8.779 −43.279 34.970 1.000 16.23ATOM 1493 O GLU 198 −8.636 −44.296 34.292 1.000 14.85 ATOM 1494 N ALA199 −8.596 −43.284 36.291 1.000 11.36 ATOM 1495 CA ALA 199 −8.233−44.489 37.022 1.000 5.99 ATOM 1496 CB ALA 199 −8.047 −44.154 38.4991.000 2.34 ATOM 1497 C ALA 199 −9.273 −45.594 36.873 1.000 7.89 ATOM1498 O ALA 199 −8.922 −46.767 36.748 1.000 16.70 ATOM 1499 N ASN 200−10.548 −45.210 36.897 1.000 13.48 ATOM 1500 CA ASN 200 −11.644 −46.15536.715 1.000 11.59 ATOM 1501 CB ASN 200 −13.007 −45.474 36.805 1.0004.12 ATOM 1502 CG ASN 200 −13.492 −45.192 38.209 1.000 11.67 ATOM 1503OD1 ASN 200 −13.045 −45.767 39.200 1.000 6.19 ATOM 1504 ND2 ASN 200−14.455 −44.276 38.330 1.000 13.74 ATOM 1505 C ASN 200 −11.505 −46.86935.366 1.000 8.88 ATOM 1506 O ASN 200 −11.667 −48.084 35.305 1.000 9.08ATOM 1507 N ASN 201 −11.208 −46.111 34.315 1.000 14.48 ATOM 1508 CA ASN201 −11.074 −46.639 32.963 1.000 14.27 ATOM 1509 CB ASN 201 −10.903−45.495 31.960 1.000 16.17 ATOM 1510 CG ASN 201 −12.221 −44.853 31.5701.000 14.25 ATOM 1511 OD1 ASN 201 −13.050 −45.436 30.871 1.000 13.77ATOM 1512 ND2 ASN 201 −12.441 −43.624 32.021 1.000 16.01 ATOM 1513 C ASN201 −9.908 −47.620 32.870 1.000 12.95 ATOM 1514 O ASN 201 −10.050−48.720 32.334 1.000 11.02 ATOM 1515 N ARG 202 −8.775 −47.207 33.4121.000 15.80 ATOM 1516 CA ARG 202 −7.571 −48.020 33.532 1.000 14.85 ATOM1517 CB ARG 202 −6.491 −47.250 34.294 1.000 17.85 ATOM 1518 CG ARG 202−5.109 −47.874 34.325 1.000 17.66 ATOM 1519 CD ARG 202 −4.141 −47.02635.143 1.000 19.69 ATOM 1520 NE ARG 202 −3.646 −45.881 34.388 1.00030.64 ATOM 1521 CZ ARG 202 −2.410 −45.407 34.412 1.000 36.54 ATOM 1522NH1 ARG 202 −1.470 −45.972 35.164 1.000 35.38 ATOM 1523 NH2 ARG 202−2.093 −44.353 33.669 1.000 23.31 ATOM 1524 C ARG 202 −7.862 −49.34434.229 1.000 6.52 ATOM 1525 O ARG 202 −7.636 −50.401 33.644 1.000 9.98ATOM 1526 N ASP 203 −8.365 −49.285 35.464 1.000 3.83 ATOM 1527 CA ASP203 −8.597 −50.500 36.237 1.000 12.72 ATOM 1528 CB ASP 203 −9.148−50.181 37.631 1.000 9.96 ATOM 1529 CG ASP 203 −8.170 −49.370 38.4581.000 16.04 ATOM 1530 OD1 ASP 203 −6.980 −49.324 38.086 1.000 18.66 ATOM1531 OD2 ASP 203 −8.584 −48.772 39.474 1.000 22.09 ATOM 1532 C ASP 203−9.548 −51.455 35.524 1.000 18.07 ATOM 1533 O ASP 203 −9.383 −52.67435.579 1.000 12.38 ATOM 1534 N LEU 204 −10.550 −50.890 34.859 1.00023.73 ATOM 1535 CA LEU 204 −11.541 −51.706 34.169 1.000 21.34 ATOM 1536CB LEU 204 −12.745 −50.872 33.727 1.000 26.39 ATOM 1537 CG LEU 204−14.123 −51.510 33.908 1.000 26.92 ATOM 1538 CD1 LEU 204 −15.079 −51.06632.809 1.000 10.26 ATOM 1539 CD2 LEU 204 −14.019 −53.027 33.942 1.00035.07 ATOM 1540 C LEU 204 −10.938 −52.392 32.948 1.000 10.84 ATOM 1541 OLEU 204 −11.212 −53.567 32.707 1.000 16.23 ATOM 1542 N GLY 205 −10.143−51.649 32.189 1.000 8.26 ATOM 1543 CA GLY 205 −9.534 −52.173 30.9841.000 6.27 ATOM 1544 C GLY 205 −8.472 −53.215 31.265 1.000 8.34 ATOM1545 O GLY 205 −8.228 −54.094 30.436 1.000 9.21 ATOM 1546 N VAL 206−7.829 −53.130 32.425 1.000 8.74 ATOM 1547 CA VAL 206 −6.833 −54.13532.796 1.000 9.33 ATOM 1548 CB VAL 206 −5.942 −53.653 33.957 1.000 16.14ATOM 1549 CG1 VAL 206 −5.020 −54.754 34.457 1.000 6.58 ATOM 1550 CG2 VAL206 −5.124 −52.445 33.514 1.000 6.33 ATOM 1551 C VAL 206 −7.526 −55.44733.154 1.000 5.34 ATOM 1552 O VAL 206 −7.118 −56.498 32.664 1.000 5.68ATOM 1553 N ALA 207 −8.564 −55.384 33.982 1.000 4.56 ATOM 1554 CA ALA207 −9.349 −56.547 34.369 1.000 8.39 ATOM 1555 CB ALA 207 −10.323−56.180 35.490 1.000 0.79 ATOM 1556 C ALA 207 −10.144 −57.160 33.2191.000 10.03 ATOM 1557 O ALA 207 −10.485 −58.346 33.261 1.000 13.69 ATOM1558 N LEU 208 −10.471 −56.382 32.193 1.000 14.72 ATOM 1559 CA LEU 208−11.278 −56.888 31.082 1.000 11.49 ATOM 1560 CB LEU 208 −12.065 −55.75530.422 1.000 12.04 ATOM 1561 CG LEU 208 −13.325 −55.317 31.175 1.00010.97 ATOM 1562 CD1 LEU 208 −13.985 −54.127 30.497 1.000 18.17 ATOM 1563CD2 LEU 208 −14.302 −56.477 31.290 1.000 17.03 ATOM 1564 C LEU 208−10.391 −57.604 30.067 1.000 6.10 ATOM 1565 O LEU 208 −10.857 −58.50229.369 1.000 15.12 ATOM 1566 N ALA 209 −9.132 −57.191 30.019 1.000 10.78ATOM 1567 CA ALA 209 −8.103 −57.815 29.203 1.000 16.00 ATOM 1568 CB ALA209 −6.827 −56.992 29.220 1.000 18.55 ATOM 1569 C ALA 209 −7.829 −59.23829.694 1.000 19.15 ATOM 1570 O ALA 209 −7.639 −60.143 28.882 1.000 13.89ATOM 1571 N GLU 210 −7.822 −59.396 31.015 1.000 9.97 ATOM 1572 CA GLU210 −7.645 −60.692 31.653 1.000 11.15 ATOM 1573 CB GLU 210 −7.535−60.520 33.168 1.000 21.07 ATOM 1574 CG GLU 210 −6.097 −60.365 33.6471.000 39.63 ATOM 1575 CD GLU 210 −5.696 −58.921 33.860 1.000 47.94 ATOM1576 OE1 GLU 210 −5.958 −58.391 34.960 1.000 64.71 ATOM 1577 OE2 GLU 210−5.097 −58.319 32.949 1.000 43.70 ATOM 1578 C GLU 210 −8.791 −61.63431.308 1.000 10.80 ATOM 1579 O GLU 210 −8.589 −62.787 30.927 1.000 10.93ATOM 1580 N GLN 211 −10.007 −61.120 31.441 1.000 10.29 ATOM 1581 CA GLN211 −11.190 −61.871 31.035 1.000 17.12 ATOM 1582 CB GLN 211 −12.443−61.052 31.363 1.000 15.73 ATOM 1583 CG GLN 211 −12.542 −60.709 32.8441.000 19.97 ATOM 1584 CD GLN 211 −12.936 −61.923 33.671 1.000 20.12 ATOM1585 OE1 GLN 211 −13.886 −62.628 33.331 1.000 17.44 ATOM 1586 NE2 GLN211 −12.218 −62.166 34.759 1.000 12.84 ATOM 1587 C GLN 211 −11.146−62.237 29.556 1.000 19.66 ATOM 1588 O GLN 211 −11.399 −63.384 29.1701.000 12.73 ATOM 1589 N VAL 212 −10.822 −61.287 28.679 1.000 17.48 ATOM1590 CA VAL 212 −10.785 −61.612 27.249 1.000 19.02 ATOM 1591 CB VAL 212−10.426 −60.369 26.415 1.000 14.47 ATOM 1592 CG1 VAL 212 −10.189 −60.74424.958 1.000 15.00 ATOM 1593 CG2 VAL 212 −11.527 −59.320 26.523 1.0008.88 ATOM 1594 C VAL 212 −9.816 −62.745 26.936 1.000 23.29 ATOM 1595 OVAL 212 −10.192 −63.735 26.294 1.000 25.62 ATOM 1596 N ARG 213 −8.557−62.645 27.361 1.000 21.16 ATOM 1597 CA ARG 213 −7.617 −63.740 27.1261.000 22.08 ATOM 1598 CB ARG 213 −6.251 −63.462 27.752 1.000 19.45 ATOM1599 CG ARG 213 −5.577 −62.178 27.300 1.000 20.41 ATOM 1600 CD ARG 213−4.621 −61.690 28.380 1.000 26.40 ATOM 1601 NE ARG 213 −3.847 −60.52727.952 1.000 29.86 ATOM 1602 CZ ARG 213 −3.556 −59.504 28.745 1.00026.00 ATOM 1603 NH1 ARG 213 −3.968 −59.485 30.007 1.000 15.34 ATOM 1604NH2 ARG 213 −2.847 −58.491 28.268 1.000 17.74 ATOM 1605 C ARG 213 −8.157−65.052 27.695 1.000 21.76 ATOM 1606 O ARG 213 −7.893 −66.138 27.1821.000 28.34 ATOM 1607 N SER 214 −8.924 −64.952 28.780 1.000 15.76 ATOM1608 CA SER 214 −9.486 −66.151 29.389 1.000 15.09 ATOM 1609 CB SER 214−10.043 −65.824 30.781 1.000 19.35 ATOM 1610 OG SER 214 −11.053 −66.74531.144 1.000 46.77 ATOM 1611 C SER 214 −10.561 −66.790 28.529 1.00015.48 ATOM 1612 O SER 214 −10.692 −68.016 28.535 1.000 24.87 ATOM 1613 NLEU 215 −11.355 −66.030 27.772 1.000 21.40 ATOM 1614 CA LEU 215 −12.367−66.673 26.938 1.000 21.52 ATOM 1615 CB LEU 215 −13.655 −65.855 26.8601.000 22.40 ATOM 1616 CG LEU 215 −14.176 −65.153 28.103 1.000 20.48 ATOM1617 CD1 LEU 215 −15.071 −63.990 27.697 1.000 27.15 ATOM 1618 CD2 LEU215 −14.931 −66.118 29.006 1.000 13.10 ATOM 1619 C LEU 215 −11.884−66.920 25.510 1.000 20.60 ATOM 1620 O LEU 215 −12.536 −67.682 24.7891.000 31.41 ATOM 1621 N LEU 216 −10.790 −66.303 25.077 1.000 21.43 ATOM1622 CA LEU 216 −10.291 −66.503 23.718 1.000 19.55 ATOM 1623 CB LEU 216−10.114 −65.148 23.021 1.000 19.47 ATOM 1624 CG LEU 216 −11.385 −64.30522.870 1.000 16.11 ATOM 1625 CD1 LEU 216 −11.095 −63.042 22.076 1.00017.60 ATOM 1626 CD2 LEU 216 −12.495 −65.108 22.211 1.000 4.00 ATOM 1627C LEU 216 −8.983 −67.283 23.688 1.000 24.37 ATOM 1628 OT1 LEU 216 −8.472−67.525 22.571 1.000 29.22 ATOM 1629 OT2 LEU 216 −8.463 −67.655 24.7581.000 19.02

In addition to the above-described determinations, a carbamate-inhibitedperhydrolase crystal was also produced and analyzed. In theseexperiments, a N-hexylcarbamate derivative of wild type perhydrolase wasused. Wild-type perhydrolase (14.5 mg in 1 mL, 67 mM NaPO4 pH 7 buffer)was titrated at room temperature with 1.25 μL aliquots of 400 mMp-nitrophenyl-N-hexylcarbamate dissolved in DMSO. Perhydrolase activitywas measured with p-nitrophenylbutyrate assay (See, Example 2), as afunction of time after each addition of the inhibitor. Several additionsover several hours were required for complete inhibition of the enzyme.After inhibition was complete, the buffer of the inhibited enzymesolution was exchanged for 10 mM HEPES pH 8.3. This solution was storedat −80° C. until used for crystallization screening experiments wereconducted as described above. The inhibitorp-nitrophenyl-N-hexylcarbamate was prepared by methods known in the art(See e.g., Hosie et al., J. Biol. Chem., 262:260-264 [1987]). Briefly,the carbamate-inhibited perhydrolase was crystallized by vapor diffusionusing the hanging drop method known in the art. A ml solution ofinhibited perhydrolase (15 mg/ml in 10 mM HEPES, pH 8.2), was mixed with4 μL of a reservoir solution (30% PEG-4,000 with 0.2 M lithium sulfateand 0.1 M Tris, pH 8.5) on a plastic coverslip, then inverted and sealedfor a well of 6×4 Linbro plate containing 0.5 ml of the reservoirsolution and allowed to equilibrate. Crystals formed within a few days.The crystals were flash frozen in liquid nitrogen and analyzed asdescribed above.

While the native octamer was determined in space group P4 with unit celldimensions:

a=98.184 b=98.184 and c=230.119 α=90.00 β=90.00 γ=90.00, this crystaldiffracted to about 2.0 Å. The carbamate-inhibited crystal grew in thespace group P1 with unit cell dimensions a=67.754, b=80.096, andc=85.974 α=104.10°, β=112.10°, and γ=97.40° and these crystals diffractto a resolution exceeding 1.0 Å.

The carbamate was bound in a manner to exploit the interactions betweenthe keto oxygen of the carbamate and residues forming the oxyanion hole,the amide N atoms of Ser 11 and Ala 55 and Asn 94 ND2. The hydrophobicside chain extends along the hydrophobic surface of the binding site outinto the surface opening between pairs of dimers in the octamerstructure. The carbamate moiety direction highlights the pivotal role ofthe S54V mutation. The hydrophobic moiety passes adjacent to the sidechain of ser 54. Mutating the serine side to valine increased thehydrophobicity, and also served as a gatekeeper to prevent hydrophilicnucleophiles (e.g., water) for competing with desired deacylatingnucleophiles. The t residues surrounding the carbamate moiety on thesame and neighboring molecules forming the extended entry are expectedto influence the selection of the optimal de-acylating nucleophile.

In addition, residues with surface-accessible side chain atoms wereidentified using the program “AreaMol,” within the CCP4 program package.Table 15-1 lists these residues. In this Table, the residue number,residue name, number of surface-accessible side chain atoms having atleast 10.0 square atoms of accessible surface area, and maximum surfacearea (square angstroms) for any side chain atom within that residue (orCA for GLY residues) in the octameric structure of perhydrolase areprovided.

TABLE 15-1 Surface-Accessible Side Chain Atoms Maximum Surface ResidueResidue Number of Accessible Area (Square Number Name Side Chain AtomsAngstroms) 1 ALA 1 15.7 3 LYS′ 2 54.10 17 VAL 1 29.5 19 VAL 1 28.0 20GLU 4 30.2 21 ASP 2 41.3 24 PRO 2 23.2 26 GLU 3 36.3 29 ALA 1 34.4 30PRP 3 32.7 31 ASP 3 50.6 32 VAL 1 27.0 39 ALA 1 27.5 40 GLN 3 38.7 41GLN 2 22.1 43 GLY 1 20.4 44 ALA 1 63.8 45 ASP 3 52.7 46 PHE 2 17.1 47GLU 3 29.6 61 ASP 3 53.1 63 PRO 3 28.0 64 THR 1 15.7 65 ASP 1 10.8 66PRO 3 33.5 67 ARG 2 20.3 69 ASN 1 11.0 72 SER 2 26.6 75 PRO 2 17.4 83PRO 2 15.1 85 ASP 1 36.80 98 ALA 1 14.60 101 ARG 4 25.0 102 ARG 1 19.9103 THR 1 43.7 104 PRO 1 17.90 105 LEU 1 10.1 113 VAL 1 17.3 116 THR 239.5 117 GLN 2 15.3 119 LEU 3 21.4 120 THR 2 34.1 122 ALA 1 38.0 123 GLY1 11.0 126 GLY 1 11.9 128 THR 2 18.2 129 TYR 1 17.6 130 PRO 3 30.2 131ALA 1 13.7 133 LYS 3 46.9 141 PRO 3 25.3 143 ALA 1 19.8 144 PRO 3 34.90146 PRO 2 24.30 148 PRO 3 24.1 151 GLN 3 35.6 152 LEU 1 12.90 155 GLU 353.0 156 GLY 1 28.9 158 GLU 3 30.3 159 GLN 4 44.9 160 LYS 2 21.5 162 THR2 25.0 163 GLU 2 23.3 165 ALA 1 23.1 169 SER 1 39.1 173 SER 2 33.3 174PHE 1 11.1 175 MET 1 18.5 176 LYS 2 21.4 178 PRO 1 12.0 179 PHE 2 14.0180 PHE 1 13.9 181 ASP 1 24.9 184 SER 1 27.0 185 VAL 1 27.5 187 SER 234.0 189 ASP 2 25.4 191 VAL 2 24.5 197 THR 2 21.6 198 GLU 3 43.5 199 ALA1 50.5 202 ARG 3 37.2 203 ASP 2 30.9 206 VAL 2 45.2 210 GLU 3 34.6 211GLN 2 19.6 213 ARG 5 30.8 214 SER 2 20.8 215 LEU 1 25.80

Example 16 Stain Removal

In this Example, experiments conducted to assess the stain removalabilities of perhydrolase are described.

Individual wells of 24 well culture plates were used to mimic conditionsfound in ordinary washing machines. Each well was filled withcommercially available detergent (e.g., Ariel [Procter & Gamble], WOB[AATCC], and WFK [WFK]), and pre-stained cloth discs cut to fit insideof each well were added. Temperature and agitation were accomplished byattaching the plate to the inside of a common laboratoryincubator/shaker. To measure bleaching effectiveness of theperhydrolase, fabric stained with tea (EMPA #167, available commerciallyfrom Test Fabrics) was used. A single cloth disc was placed in eachwell, and 1 ml of detergent liquid, containing enzyme, ester substrate,and peroxide was added. After agitation at 100-300 rpm @ 20-60° C., thefabric discs were removed, rinsed with tap water, and allowed to dryovernight. The reflectance of each individual cloth disc was measured,and plotted as an “L” value. These results are provided in FIG. 21,which shows that the addition of the perhydrolase of the presentinvention to the detergent consistently provides a greater degree ofbleaching than the detergents alone. In this Figure, “E” indicates theresults for each of the detergents tested in combination with theperhydrolase of the present invention.

Example 17 Cotton Bleaching

In this Example, experiments to assess the use of the perhydrolase ofthe present invention for bleaching of cotton fabrics are described.

In these experiments, six cotton swatches per canister were treated at55° C. for 60 minutes in a Launder-O-meter. The substrates used in theseexperiments were: 3 (3″×3″) 428U and 3 (3″×3″) 400U per experiments. Twodifferent types of 100% unbleached cotton fabrics from Testfabrics weretested (style 428U (desized but not bleached army carded cotton sateen);and style 400U (desized but not bleached cotton print cloth). The liquorratio was about 26 to 1 (˜7.7 g fabric/˜200 ml volume liquor). Theperhydrolase enzyme was tested at 12.7 mgP/ml, with ethyl acetate (3%(v/v)), hydrogen peroxide (1500 ppm), and Triton X-100 (0.001%), in asodium phosphate buffer (100 mM) for pH 7 and pH 8; as well as in asodium carbonate (100 mM) buffer, for pH 9 and pH 10.

Bleaching effects were quantified with total color difference by taking4 CIE L*a*b* values per each swatch before and after the treatmentsusing a Chroma Meter CR-200 (Minolta), and total color difference of theswatches after the treatments were calculated according to thefollowing:Total color difference

$\left( {\Delta\; E} \right) = \sqrt{\left( {{\Delta\; L^{2}} + {\Delta\; a^{2}} + {\Delta\; b^{2}}} \right)}$(where ΔL, Δa, Δb, are differences in CIE L*, CIE a*, and CIE b* valuesrespectively before and after the treatments).

Higher ΔE values indicate greater bleaching effects. The results (See,FIG. 22) indicated that the perhydrolase showed significantly improvedbleaching effects on both types of 100% cotton fabrics at pH 7 and pH 8under the conditions tested.

It was also observed that high amounts of motes (e.g., pigmented spots)disappeared on the enzyme treated substrates.

Example 18 Linen Bleaching

In this Example, experiments conducted to assess the linen bleachingcapability of the perhydrolase of the present invention are described.The same methods and conditions as describe above for cotton testing (inExample 17) were used to test linen swatches. As indicated above,experiments were conduction in a Launder-O-meter using a linen fabric(linen suiting, Style L-53; Testfabrics).

In these experiments, 3 (4″×4″) linen swatches were treated with 12.7mgP/ml of the perhydrolase enzyme with ethyl acetate (3% v/v), hydrogenperoxide (1200 ppm), and Triton X-100 (0.001%), in a sodium phosphatebuffer (100 mM) for pH 7 and pH 8. The bleaching effects were calculatedas described above in Example 17. FIG. 23 provides a graph showing thebleaching effects of the perhydrolase of the present invention tested atpH 7 and pH 8 on linen.

Example 19 Detergent Compositions

In the following Example, various detergent compositions areexemplified. In these formulations, the enzymes levels are expressed bypure enzyme by weight of the total composition and unless otherwisespecified, the detergent ingredients are expressed by weight of thetotal compositions. The abbreviated component identifications thereinhave the following meanings:

-   LAS: Sodium linear C₁₁₋₁₃ alkyl benzene sulfonate.-   TAS: Sodium tallow alkyl sulfate.-   CxyAS: Sodium C_(1x)-C_(1y) alkyl sulfate.-   CxyEz: C_(1x)-C_(1y) predominantly linear primary alcohol condensed    with an average of z moles of ethylene oxide.-   CxyAEzS: C_(1x)-C_(1y) sodium alkyl sulfate condensed with an    average of z moles of ethylene oxide. Added molecule name in the    examples.-   Nonionic: Mixed ethoxylated/propoxylated fatty alcohol e.g. Plurafac    LF404 being an alcohol with an average degree of ethoxylation of 3.8    and an average degree of propoxylation of 4.5.-   QAS: R₂.N+(CH₃)₂(C₂H₄OH) with R₂=C₁₂-C₁₄.-   Silicate: Amorphous Sodium Silicate (SiO₂:Na₂O ratio=1.6-3.2:1).-   Metasilicate: Sodium metasilicate (SiO₂:Na₂O ratio=1.0).-   Zeolite A: Hydrated Aluminosilicate of formula    Na₁₂(AlO₂SiO₂)₁₂.27H₂O-   SKS-6: Crystalline layered silicate of formula δ-Na₂Si₂O₅.-   Sulphate: Anhydrous sodium sulphate.-   STPP: Sodium Tripolyphosphate.-   MA/AA: Random copolymer of 4:1 acrylate/maleate, average molecular    weight about 70,000-80,000.-   AA: Sodium polyacrylate polymer of average molecular weight 4,500.-   Polycarboxylate: Copolymer comprising mixture of carboxylated    monomers such as acrylate, maleate and methyacrylate with a MW    ranging between 2,000-80,000 such as Sokolan commercially available    from BASF, being a copolymer of acrylic acid, MW4,500.-   BB1: 3-(3,4-Dihydroisoquinolinium)propane sulfonate-   BB2 1-(3,4-dihydroisoquinolinium)-decane-2-sulfate-   PB1: Sodium perborate monohydrate.-   PB4: Sodium perborate tetrahydrate of nominal formula NaBO₃.4H₂O.-   Percarbonate: Sodium percarbonate of nominal formula 2Na₂CO₃.3H₂O₂.-   TAED: Tetraacetyl ethylene diamine.-   NOBS: Nonanoyloxybenzene sulfonate in the form of the sodium salt.-   DTPA: Diethylene triamine pentaacetic acid.-   HEDP: 1,1-hydroxyethane diphosphonic acid.-   DETPMP: Diethyltriamine penta(methylene)phosphonate, marketed by    Monsanto under the Trade name Dequest 2060.-   EDDS: Ethylenediamine-N,N′-disuccinic acid, (S,S) isomer in the form    of its sodium salt-   Diamine: Dimethyl aminopropyl amine; 1,6-hezane diamine; 1,3-propane    diamine; 2-methyl-1,5-pentane diamine; 1,3-pentanediamine;    1-methyl-diaminopropane.-   DETBCHD 5,12-diethyl-1,5,8,12-tetraazabicyclo[6,6,2]hexadecane,    dichloride, Mn(II) salt-   PAAC: Pentaamine acetate cobalt(III) salt.-   Paraffin: Paraffin oil sold under the tradename Winog 70 by    Wintershall.-   Paraffin Sulfonate: A Paraffin oil or wax in which some of the    hydrogen atoms have been replaced by sulfonate groups.-   Aldose oxidase: Oxidase enzyme sold under the tradename Aldose    Oxidase by Novozymes A/S-   Galactose oxidase: Galactose oxidase from Sigma-   Protease: Proteolytic enzyme sold under the tradename Savinase,    Alcalase, Everlase by Novo Nordisk A/S, and the following from    Genencor International, Inc: “Protease A” described in U.S. Pat. No.    RE 34,606 in FIGS. 1A, 1B, and 7, and at column 11, lines 11-37;    “Protease B” described in U.S. Pat. No. 5,955,340 and U.S. Pat. No.    5,700,676 in FIGS. 1A, 1B and 5, as well as Table 1; and “Protease    C” described in U.S. Pat. No. 6,312,936 and U.S. Pat. No. 6,482,628    in FIGS. 1-3 [SEQ ID 3], and at column 25, line 12, “Protease D”    being the variant 101/G103A/104I/159/D232V/236H/245R/248D/252K (BPN′    numbering) described in WO 99/20723.-   Amylase: Amylolytic enzyme sold under the tradename Purafact Ox    Am^(R) described in WO 94/18314, WO96/05295 sold by Genencor;    Natalase®, Termamyl®, Fungamyl® and Duramyl®, all available from    Novozymes A/S.-   Lipase: Lipolytic enzyme sold under the tradename Lipolase Lipolase    Ultra by Novozymes A/S and Lipomax by Gist-Brocades.-   Cellulase: Cellulytic enzyme sold under the tradename Carezyme,    Celluzyme and/or Endolase by Novozymes A/S.-   Pectin Lyase: Pectaway® and Pectawash® available from Novozymes A/S.-   PVP: Polyvinylpyrrolidone with an average molecular weight of 60,000-   PVNO: Polyvinylpyridine-N-Oxide, with an average molecular weight of    50,000.-   PVPVI: Copolymer of vinylimidazole and vinylpyrrolidone, with an    average molecular weight of 20,000.-   Brightener 1: Disodium 4,4′-bis(2-sulphostyryl)biphenyl.-   Silicone antifoam: Polydimethylsiloxane foam controller with    siloxane-oxyalkylene copolymer as dispersing agent with a ratio of    said foam controller to said dispersing agent of 10:1 to 100:1.-   Suds Suppressor: 12% Silicone/silica, 18% stearyl alcohol, 70%    starch in granular form.-   SRP 1: Anionically end capped poly esters.-   PEG X: Polyethylene glycol, of a molecular weight of x.-   PVP K60®: Vinylpyrrolidone homopolymer (average MW 160,000)-   Jeffamine® ED-2001: Capped polyethylene glycol from Huntsman-   Isachem® AS: A branched alcohol alkyl sulphate from Enichem-   MME PEG (2000): Monomethyl ether polyethylene glycol (MW 2000) from    Fluka Chemie AG.-   DC3225C: Silicone suds suppresser, mixture of Silicone oil and    Silica from Dow Corning.-   TEPAE: Tetreaethylenepentaamine ethoxylate.-   BTA: Benzotriazole.-   Betaine: (CH₃)₃N⁺CH₂COO⁻-   Sugar: Industry grade D-glucose or food grade sugar-   CFAA: C₁₂-C₁₄ alkyl N-methyl glucamide-   TPKFA: C₁₂-C₁₄ topped whole cut fatty acids.-   Clay: A hydrated aluminumu silicate in a general formula    Al₂O₃SiO₂.xH₂O. Types: Kaolinite, montmorillonite, atapulgite,    illite, bentonite, halloysite.-   MCAEM: Esters in the formula of R¹O_(x)[(R²)_(m)(R³)_(n)]_(p)-   pH: Measured as a 1% solution in distilled water at 20° C.

Example 20 Liquid Laundry Detergents

The following liquid laundry detergent compositions of the presentinvention are prepared.

I II III IV V LAS 18.0 — 6.0 — — C ₁₂-C₁₅ AE_(1.8)S — 2.0 8.0 11.0 5.0C₈-C₁₀ propyl dimethyl 2.0 2.0 2.0 2.0 1.0 amine C₁₂-C₁₄ alkyl dimethyl— — — — 2.0 amine oxide C₁₂-C₁₅ AS — 17.0 — 7.0 8.0 CFAA — 5.0 4.0 4.03.0 C₁₂-C₁₄ Fatty alcohol 12.0 6.0 1.0 1.0 1.0 ethoxylate C₁₂-C₁₈ Fattyacid 11.0 11.0 4.0 4.0 3.0 Citric acid (anhydrous) 5.0 1.0 3.0 3.0 2.0DETPMP 1.0 1.0 1.0 1.0 0.5 Monoethanolamine 11.0 8.0 5.0 5.0 2.0 Sodiumhydroxide 1.0 1.0 2.5 1.0 1.5 Percarbonate — 3.5 — 2.5 — Propanediol12.7 14.5 13.1 10. 8.0 Ethanol 1.8 1.8 4.7 5.4 1.0 Pectin Lyase — — —0.005 — Amylase — 0.002 — — Cellulase — — 0.0002 0.0001 Lipase 0.1 — 0.1— 0.1 Protease A 0.05 0.3 0.055 0.5 0.2 Aldose Oxidase 0.03 — 0.3 —0.003 PAAC 0.01 0.01 — — — DETBCHD — — 0.02 0.01 — SRP1 0.5 0.5 — 0.30.3 Boric acid 2.4 2.4 2.8 2.8 2.4 Sodium xylene sulfonate — — 3.0 — —DC 3225C 1.0 1.0 1.0 1.0 1.0 2-butyl-octanol 0.03 0.04 0.04 0.03 0.03DTPA 0.5 0.4 0.35 0.28 0.4 Brightener 1 0.18 0.10 0.11 — — Perhydrolase0.05 0.3 0.08 0.5 0.2 MCAEM 3.0 8.0 12.0 1.5 4.8 (C₁₂-C₁₃ E _(6.5)Acetate) Balance to 100% perfume/dye and/or water

Example 21 Hand-Dish Liquid Detergent Compositions

The following hand dish liquid detergent compositions of the presentinvention are prepared.

I II III IV V VI C₁₂-C₁₅AE_(1.8)S 30.0 28.0 25.0 — 15.0 10.0 LAS — — —5.0 15.0 12.0 Paraffin Sulfonate — — — 20.0 — — C₁₀-C₁₈ Alkyl 5.0 3.07.0 — — — Dimethyl Amine Oxide Betaine 3.0 — 1.0 3.0 1.0 — C₁₂ poly-OHfatty — — — 3.0 — 1.0 acid amide C₁₄ poly-OH fatty — 1.5 — — — — acidamide C₁₁E₉ 2.0 — 4.0 — — 20.0 DTPA — — — — 0.2 — Tri-sodium Citrate0.25 — — 0.7 — — dihydrate Diamine 1.0 5.0 7.0 1.0 5.0 7.0 MgCl₂ 0.25 —— 1.0 — — Protease A 0.02 0.01 0.02 0.01 0.02 0.05 Amylase 0.001 — —0.002 — 0.001 Aldose Oxidase 0.03 — 0.02 — 0.05 — Sodium Cumene — — —2.0 1.5 3.0 Sulphonate PAAC 0.01 0.01 0.02 — — — DETBCHD — — — 0.01 0.020.01 PB1 1.5 2.8 1.2 — — — Perhydrolase 0.02 0.01 0.03 0.01 0.02 0.05MCAEM 3.4 2.8 4.0 2.6 4.6 6.8 (C₁₄-C₁₅E₇ Acetate) Balance to 100%perfume/dye and/or waterThe pH of Compositions (I)-(VI) is about 8 to about 11

Example 22 Liquid Automatic Dishwashing Detergent

The following liquid automatic dishwashing detergent compositions of thepresent are prepared.

I II III IV V STPP 16 16 18 16 16 Potassium Sulfate — 10 8 — 10 1,2propanediol 6.0 0.5 2.0 6.0 0.5 Boric Acid 4.0 3.0 3.0 4.0 3.0 CaCl₂dihydrate 0.04 0.04 0.04 0.04 0.04 Nonionic 0.5 0.5 0.5 0.5 0.5 ProteaseB 0.03 0.03 0.03 0.03 0.03 Amylase 0.02 — 0.02 0.02 — Aldose Oxidase —0.15 0.02 — 0.01 Galactose Oxidase — — 0.01 — 0.01 PAAC 0.01 — — 0.01 —DETBCHD — 0.01 — — 0.01 Perhydrolase 0.1 0.03 0.05 0.03 0.06 MCAEM 5.03.0 12.0 8.0 1.0 (C₁₄-C₁₅E₁₂ Acetate) Balance to 100% perfume/dye and/orwater

Example 23 Laundry Compositions

The following laundry compositions of present invention, which may be inthe form of granules or tablet, are prepared.

Base Product I II III IV V C₁₄-C₁₅AS or TAS 8.0 5.0 3.0 3.0 3.0 LAS 8.0— 8.0 — 7.0 C₁₂-C₁₅AE₃S 0.5 2.0 1.0 — — C₁₂-C₁₅E₅ or E₃ 2.0 — 5.0 2.02.0 QAS — — — 1.0 1.0 Zeolite A 20.0 18.0 11.0 — 10.0 SKS-6 (dry add) —— 9.0 — — MA/AA 2.0 2.0 2.0 — — AA — — — — 4.0 3Na Citrate 2H₂O — 2.0 —— — Citric Acid (Anhydrous) 2.0 — 1.5 2.0 — DTPA 0.2 0.2 — — — EDDS — —0.5 0.1 — HEDP — — 0.2 0.1 — PB1 3.0 4.8 — — 4.0 Percarbonate — — 3.85.2 — NOBS 1.9 — — — — NACA OBS — — 2.0 — — TAED 0.5 2.0 2.0 5.0 1.00BB1 0.06 — 0.34 — 0.14 BB2 — 0.14 — 0.20 — Anhydrous Na Carbonate 15.018.0 8.0 15.0 15.0 Sulfate 5.0 12.0 2.0 17.0 3.0 Silicate — 1.0 — — 8.0Protease B 0.033 0.033 — — — Protease C — — 0.033 0.046 0.033 Lipase —0.008 — — — Amylase 0.001 — — — 0.001 Cellulase — 0.0014 — — — PectinLyase 0.001 0.001 0.001 0.001 0.001 Aldose Oxidase 0.03 — 0.05 — — PAAC— 0.01 — — 0.05 Perhydrolase 0.03 0.05 1.0 0.06 0.1 MCAEM** 2.0 5.0 12.03.5 6.8 Balance to 100% Moisture and/or Minors* Perfume/Dye,Brightener/SRP1/Na Carboxymethylcellulose/Photobleach/MgSO₄/PVPVI/Sudssuppressor/High Molecular PEG/Clay. **MCAEM is selected from the groupconsisting of C ₉-C₁₁E_(2.5) Acetate, [C₁₂H₂₅N(CH₃)(CH₂CH₂OAc)₂]⁺ Cl⁻,(CH₃)₂NCH₂CH₂OCH₂CH₂OAc, or mixtures thereof..

Example 24 Liquid Laundry Detergents

The following liquid laundry detergent formulations of the presentinvention are prepared.

I I II III IV V LAS 11.5 11.5 9.0 — 4.0 — C₁₂-C₁₅AE_(2.85)S — — 3.0 18.0— 16.0 C₁₄-C₁₅E_(2.5)S 11.5 11.5 3.0 — 16.0 — C₁₂-C₁₃E₉ — — 3.0 2.0 2.01.0 C₁₂-C₁₃E₇ 3.2 3.2 — — — — CFAA — — — 5.0 — 3.0 TPKFA 2.0 2.0 — 2.00.5 2.0 Citric Acid 3.2 3.2 0.5 1.2 2.0 1.2 (Anhydrous) Ca formate 0.10.1 0.06 0.1 — — Na formate 0.5 0.5 0.06 0.1 0.05 0.05 Na Culmene 4.04.0 1.0 3.0 1.2 — Sulfonate Borate 0.6 0.6 — 3.0 2.0 3.0 Na hydroxide6.0 6.0 2.0 3.5 4.0 3.0 Ethanol 2.0 2.0 1.0 4.0 4.0 3.0 1,2 Propanediol3.0 3.0 2.0 8.0 8.0 5.0 Mono- 3.0 3.0 1.5 1.0 2.5 1.0 ethanolamine TEPAE2.0 2.0 — 1.0 1.0 1.0 PB1 — 4.5 — 2.8 — Protease A 0.03 0.03 0.01 0.030.02 0.02 Lipase — — — 0.002 — — Amylase — — — — 0.002 — Cellulase — — —— — 0.0001 Pectin Lyase 0.005 0.005 — — — Aldose Oxidase 0.05 — — 0.05 —0.02 Galactose — 0.04 oxidase Perhydrolase 0.03 0.05 0.01 0.03 0.08 0.02MCAEM 3.2 4.6 1.8 3.5 6.2 2.8 (C₁₂-C₁₂E₆ Acetate) PAAC 0.03 0.03 0.02 —— — DETBCHD — — — 0.02 0.01 — SRP 1 0.2 0.2 — 0.1 — — DTPA — — — 0.3 — —PVNO — — — 0.3 — 0.2 Brightener 1 0.2 0.2 0.07 0.1 — — Silicone 0.040.04 0.02 0.1 0.1 0.1 antifoam Balance to 100% perfume/dye, and/or water

Example 25 Compact High-Density Dishwashing Detergents

The following compact high density dishwashing detergent of the presentinvention are prepared:

I II III IV V VI STPP — 45.0 45.0 — — 40.0 3Na Citrate 2H₂O 17.0 — —50.0 40.2 — Na Carbonate 17.5 14.0 20.0 — 8.0 33.6 Bicarbonate — — —26.0 — — Silicate 15.0 15.0 8.0 — 25.0 3.6 Metasilicate 2.5 4.5 4.5 — —— PB1 — — 4.5 — — — PB4 — — — 5.0 — — Percarbonate — — — — — 4.8 BB1 —0.1 0.1 — 0.5 — BB2 0.2 0.05 — 0.1 — 0.6 Nonionic 2.0 1.5 1.5 3.0 1.95.9 HEDP 1.0 — — — — — DETPMP 0.6 — — — — — PAAC 0.03 0.05 0.02 — — —Paraffin 0.5 0.4 0.4 0.6 — — Protease B 0.072 0.053 0.053 0.026 0.0590.01 Amylase 0.012 — 0.012 — 0.021 0.006 Lipase — 0.001 — 0.005 — —Pectin Lyase 0.001 0.001 0.001 — — — Aldose Oxidase 0.05 0.05 0.03 0.010.02 0.01 Perhydrolase 0.072 0.053 0.053 0.026 0.059 0.01 MCAEM 3.5 2.81.6 7.5 4.2 0.8 (C₁₂-C₁₃E_(6.5) Acetate) BTA 0.3 0.2 0.2 0.3 0.3 0.3Polycarboxylate 6.0 — — — 4.0 0.9 Perfume 0.2 0.1 0.1 0.2 0.2 0.2Balance to 100% Moisture and/or Minors* *Brightener/Dye/SRP1/NaCarboxymethylcellulose/Photobleach/MgSO₄/PVPVI/Suds suppressor/HighMolecular PEG/Clay. The pH of compositions (I) through (VI) is fromabout 9.6 to about 11.3.

Example 26 Tablet Detergent Compositions

The following tablet detergent compositions of the present invention areprepared by compression of a granular dishwashing detergent compositionat a pressure of 13KN/cm² using a standard 12 head rotary press.

I II III IV V VI VII VIII STPP — 48.8 44.7 38.2 — 42.4 46.1 36.0 3NaCitrate 2H₂O 20.0 — — — 35.9 — — — Na Carbonate 20.0 5.0 14.0 15.4 8.023.0 20.0 28.0 Silicate 15.0 14.8 15.0 12.6 23.4 2.9 4.3 4.2 Lipase0.001 — 0.01 — 0.02 — — — Protease B 0.042 0.072 0.042 0.031 — — — —Protease C — — — — 0.052 0.023 0.023 0.029 Perhydrolase 0.01 0.08 0.050.04 0.052 0.023 0.023 0.029 MCAEM 2.8 6.5 4.5 3.8 4.6 2.8 2.8 2.8(C₁₂-C₁₃E_(6.5) Acetate) Amylase 0.012 0.012 0.012 — 0.015 — 0.017 0.002Pectin Lyase 0.005 — — 0.002 — — — — Aldose Oxidase — 0.03 — 0.02 0.02 —0.03 — PB1 — — 3.8 — 7.8 — — 8.5 Percarbonate 6.0 — — 6.0 — 5.0 — — BB10.2 — 0.5 — 0.3 0.2 — — BB2 — 0.2 — 0.5 — — 0.1 0.2 Nonionic 1.5 2.0 2.02.2 1.0 4.2 4.0 6.5 PAAC 0.01 0.01 0.02 — — — — — DETBCHD — — — 0.020.02 — — — TAED — — — — — 2.1 — 1.6 HEDP 1.0 — — 0.9 — 0.4 0.2 — DETPMP0.7 — — — — — — — Paraffin 0.4 0.5 0.5 0.5 — — 0.5 — BTA 0.2 0.3 0.3 0.30.3 0.3 0.3 — Polycarboxylate 4.0 — — — 4.9 0.6 0.8 — PEG 400-30,000 — —— — — 2.0 — 2.0 Glycerol — — — — — 0.4 — 0.5 Perfume — — — 0.05 0.2 0.20.2 0.2 Balance to 100% Moisture and/or Minors* *Brightener/Dye/SRP1/NaCarboxymethylcellulose/Photobleach/MgSO₄/PVPVI/Suds suppressor/HighMolecular PEG/Clay. The pH of Compositions (I) through 7(VIII) is fromabout 10 to about 11.5. The tablet weight of Compositions 7(I) through7(VIII) is from about 20 grams to about 30 grams.

Example 27 Liquid Hard Surface Cleaning Detergents

The following liquid hard surface cleaning detergent compositions of thepresent invention are prepared.

I II III IV V VI VII C₉-C₁₁E₅ 2.4 1.9 2.5 2.5 2.5 2.4 2.5 C₁₂-C₁₄E₅ 3.62.9 2.5 2.5 2.5 3.6 2.5 C₇-C₉E₆ — — — — 8.0 — — C₁₂-C₁₄E₂₁ 1.0 0.8 4.02.0 2.0 1.0 2.0 LAS — — — 0.8 0.8 — 0.8 Sodium culmene sulfonate 1.5 2.6— 1.5 1.5 1.5 1.5 Isachem ® AS 0.6 0.6 — — — 0.6 — Na₂CO₃ 0.6 0.13 0.60.1 0.2 0.6 0.2 3Na Citrate 2H₂O 0.5 0.56 0.5 0.6 0.75 0.5 0.75 NaOH 0.30.33 0.3 0.3 0.5 0.3 0.5 Fatty Acid 0.6 0.13 0.6 0.1 0.4 0.6 0.4 2-butyloctanol 0.3 0.3 — 0.3 0.3 0.3 0.3 PEG DME-2000 ® 0.4 — 0.3 0.35 0.5 — —PVP 0.3 0.4 0.6 0.3 0.5 — — MME PEG (2000) ® — — — — — 0.5 0.5Jeffamine ® ED-2001 — 0.4 — — 0.5 — — PAAC — — — 0.03 0.03 0.03 —DETBCHD 0.03 0.05 0.05 — — — — Protease B 0.07 0.05 0.05 0.03 0.06 0.010.04 Amylase 0.12 0.01 0.01 — 0.02 — 0.01 Lipase — 0.001 — 0.005 — 0.005— Perhydrolase 0.07 0.05 0.08 0.03 0.06 0.01 0.04 MCAEM (C₁₂-C₁₅E₈ 3.55.6 4.8 5.3 3.6 8.0 4.7 Acetate) Pectin Lyase 0.001 — 0.001 — — — 0.002PB1 — 4.6 — 3.8 — — — Aldose Oxidase 0.05 — 0.03 — 0.02 0.02 0.05Balance to 100% perfume/dye, and/or water The pH of Compositions (I)through (VII) is from about 7.4 to about 9.5.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

Having described the preferred embodiments of the present invention, itwill appear to those ordinarily skilled in the art that variousmodifications may be made to the disclosed embodiments, and that suchmodifications are intended to be within the scope of the presentinvention.

Those of skill in the art readily appreciate that the present inventionis well adapted to carry out the objects and obtain the ends andadvantages mentioned, as well as those inherent therein. Thecompositions and methods described herein are representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. It is readily apparent to oneskilled in the art that varying substitutions and modifications may bemade to the invention disclosed herein without departing from the scopeand spirit of the invention.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

What is claimed is:
 1. A cleaning, bleaching or disinfectingcomposition, comprising an isolated perhydrolase, which is a homolog ofM. smegmatis perhydrolase and is a SGNH-hydrolase family polypeptide,which comprises: a) the motifs GDSL, GRTT, and GAND; and b) the aminoacid residues corresponding to L6, W14, W34, L38, R56, D62, L74, L78,H81, P83, M90, K97, G110, L114, L135, F180, G205, S11, D192, and H195 ofSEQ ID NO: 2; wherein said perhydrolase exhibits: i) perhydrolysisactivity that is at least about 0.2 compared to the perhydrolysisactivity exhibited by M. smegmatis perhydrolase; and ii) a perhydrolysisto hydrolysis ratio that is greater than 1; wherein said perhydrolysisactivity and said perhydrolysis to hydrolysis ratio is determined usingthe perhydrolysis and hydrolysis assays as described in Example
 2. 2.The composition of claim 1, wherein said perhydrolase has at least 90%amino acid sequence identity to the amino acid sequence set forth in SEQID NO:
 97. 3. The composition of claim 2, wherein said perhydrolase isMesorhizobium loti Mlo I (Q98MY5) and comprises the amino acid sequenceas set out in SEQ ID NO:
 97. 4. A cleaning composition according to anyone of claims 1, comprising: a) at least 0.0001 weight percent of saidperhydrolase; b) a molecule comprising an ester moiety; and c)optionally, an adjunct ingredient.
 5. The cleaning composition of claim4, wherein said composition comprises: a) at least 0.0001 weight percentof said perhydrolase; b) a material selected from the group consistingof: a peroxygen source, hydrogen peroxide and mixtures thereof, saidperoxygen source being selected from the group consisting of: i) aper-salt; ii) an organic peroxyacid; iii) urea hydrogen peroxide; iv) acarbohydrate and carbohydrate oxidase mixture, and v) mixtures thereof;c) from about 0.01 to about 50 weight percent of a molecule comprisingan ester moiety; and d) optionally, an adjunct ingredient.
 6. Thecleaning composition of claim 4, wherein said adjunct ingredient isselected from the group consisting of: surfactants, builders, chelatingagents, dye transfer inhibiting agents, deposition aids, dispersants,enzymes, and enzyme stabilizers, catalytic materials, bleach activators,bleach boosters, preformed peracids, polymeric dispersing agents, claysoil removal/anti-redeposition agents, brighteners, suds suppressors,dyes, perfumes, structure elasticizing agents, fabric softeners,carriers, hydrotropes, processing aids, pigments and mixtures thereof.7. The cleaning composition of claim 5, wherein: a) said per-salt isselected from the group consisting of alkalimetal perborate, alkalimetalpercarbonate, alkalimetal perphosphates, alkalimetal persulphates andmixtures thereof; b) said carbohydrate is selected from the groupconsisting of mono-carbohydrates, di-carbohydrates, tri-carbohydrates,oligo-carbohydrates and mixtures thereof; c) said carbohydrate oxidaseis selected from the group consisting of aldose oxidase (IUPACclassification EC1.1.3.9), galactose oxidase (IUPAC classificationEC1.1.3.9), cellobiose oxidase (IUPAC classification EC1.1.3.25),pyranose oxidase (IUPAC classification EC1.1.3.10), sorbose oxidase(IUPAC classification EC1.1.3.11) hexose oxidase (IUPAC classificationEC1.1.3.5), glucose oxidase (IUPAC classification EC1.1.3.4) andmixtures thereof; and d) said molecule comprising an ester moiety hasthe formula:R¹O_(x)[(R²)_(m)(R³)_(n)]_(p) i) wherein R¹ is a moiety selected fromthe group consisting of H, substituted or unsubstituted alkyl,heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, andheteroaryl; ii) each R² is an alkoxylate moiety; iii) R³ is anester-forming moiety having the formula: R⁴CO— wherein R⁴ is H, alkyl,alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; iv)x is 1 when R¹ is H; when R¹ is not H, x is an integer that is equal toor less than the number of carbons in R¹; v) p is an integer that isequal to or less than x; vi) m is an integer from 0 to 50; and vii) n isat least
 1. 8. The cleaning composition of claim 7, wherein: a) R¹ is anC₂-C₃₂ substituted or unsubstituted alkyl or heteroalkyl moiety; b) eachR² is independently an ethoxylate or propoxylate moiety; c) m is aninteger from 1 to 12; and d) R³ is an ester-forming moiety having theformula: R⁴CO— wherein R⁴ is: i) a substituted or unsubstituted alkyl,alkenyl or alkynyl moiety comprising from 1 to 22 carbon atoms; or ii) asubstituted or unsubstituted aryl, alkylaryl, alkylheteroaryl orheteroaryl moiety comprising from 4 to 22 carbon atoms.
 9. The cleaningcomposition of claim 4, wherein the molecule comprising the ester moietyhas the formula:R¹O_(x)[(R²)_(m)(R³)_(n)]_(p) wherein: a) R¹ is H or a moiety thatcomprises a primary, secondary, tertiary or quaternary amine moiety,said R¹ moiety that comprises an amine moiety being selected from thegroup consisting of substituted or unsubstituted alkyl, heteroalkyl,alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; b)each R² is an alkoxylate moiety; c) R³ is an ester-forming moiety havingthe formula: R⁴CO— wherein R⁴ may be H, substituted or unsubstitutedalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, andheteroaryl; d) x is 1 when R¹ is H; when R¹ is not H, x is an integerthat is equal to or less than the number of carbons in R¹; e) p is aninteger that is equal to or less than x f) m is an integer from 0 to 12;and g) n is at least 1; and optionally wherein said molecule comprisingan ester moiety has a weight average molecular weight of less than600,000 Daltons.
 10. The cleaning composition of claim 4, wherein saidmolecule comprising an ester moiety is selected from triacetin, ethyleneglycol diacetate and propylene glycol diacetate.
 11. The composition ofclaim 1, wherein said composition is a bleaching or disinfectingcomposition, further comprising at least one additional enzyme or enzymederivative selected from the group consisting of: proteases, amylases,lipases, mannanases, pectinases, cutinases, oxidoreductases,endoglycosidases, lysozyme, bacterial cell wall degrading enzymes,fungal cell wall degrading enzymes, hemicellulases, and cellulases. 12.A method of disinfecting and/or bleaching a surface or an article, themethod comprising contacting said surface or article with thecomposition of claim 1, optionally wherein the surface or article isselected from textiles, hard surfaces, paper, pulp, hair, teeth, skin,medical devices, medical equipment, industrial equipment and fermenters.13. A method of cleaning comprising the steps of: a) contacting asurface and/or an article comprising a fabric with a cleaningcomposition of claim 1; and b) optionally washing and/or rinsing thesurface or material.